Methods for treating mendelian disorders of the epigenetic machinery

ABSTRACT

A method is provided for treating a Mendelian disorder of the epigenetic machinery in a subject in need thereof. In particular, the method comprises administering a therapeutically effective amount of an agent that restores balance between open and closed chromatin states at one or more target genes, wherein the agent that restores balance between open and closed chromatin states at one or more target genes is an agent that ameliorates the effect of a defective gene encoding a component of the epigenetic machinery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 National Stage Entry ofInternational Application No. PCT/US15/033182 having an internationalfiling date of May 29, 2015, which claims the benefit of U.S.Provisional Application No. 62/005,607, filed May 30, 2014, the contentsof which are incorporated herein by reference in their entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“111232-00410_ST25.txt”. The sequence listing is 2,016 bytes in size,and was created on May 22, 2015. It is hereby incorporated herein byreference in its entirety.

BACKGROUND

Mendelian disorders of the epigenetic machinery are a newly delineatedgroup of multiple congenital anomaly and intellectual disabilitysyndromes resulting from mutations in genes encoding components of theepigenetic machinery. The gene products affected in these inheritedconditions act in trans and are expected to have widespread epigeneticconsequences. The DNA methylation machinery and the histone machineryaffect the expression of many genes in trans (Berdasco & Esteller (2013)Hum. Genet. 132:359-83; Wolffe (1994) Trends Biochem. Sci. 19:240-44).Within this group, genetic mutations may occur in writers, erasers, orreaders of epigenetic marks. The writers of epigenetic marks, which canbe conceptualized as a set of highlighters, place the appropriatemodifications on particular regions of the genome based on the celltype, developmental stage, and metabolic state of the cell. These marks“highlight” individual regions for use or disuse depending on whetherthe mark favors a more open or more closed chromatin state. The erasersof epigenetic marks remove these same marks, favoring the oppositechromatin states. The readers of epigenetic marks recognize andinterpret particular marks locally and give cells a mechanism forkeeping track of the overall chromatin state.

Mendelian disorders of the histone machinery have been described forwriters, erasers, readers, and chromatin remodelers. The histone writerand eraser system is unique because it involves opposing players thatmust achieve a balance of activity and subsequently of histone marks atparticular target genes in any given cell state. This idea isillustrated by Kabuki syndrome (KS), which can be caused by a defect ineither a writer or an eraser. KS is an autosomal dominant or X-linkedintellectual disability syndrome with specific dysmorphic features,including a flattened facial appearance with characteristic eyesexhibiting long palpebral fissures, eversion of the lower lids, highlyarched eyebrows, and long eyelashes, as well as short stature.

KS is caused by heterozygous loss-of-function mutations in either of twogenes with complementary functions, lysine-specific methyltransferase 2D(KMT2D) on human chromosome 12 (also known as mixed lineage leukemia 2or MLL2; Ng et al. (2010) Nat. Genet. 42:790-3) or lysine-specificdemethylase 6A (KDM6A) on human chromosome X (Lederer et al. (2012) Am.J. Hum. Genet. 90:119-24) (FIG. 2). KMT2D is a methyltransferase thatadds a trimethylation mark to H3K4 (H3K4me3, an open chromatin mark)while KDM6A is a demethylase that removes trimethylation from histone 3lysine 27 (H3K27me3, a closed chromatin mark). Both genes facilitate theopening of chromatin and promote gene expression (Ng et al. (2010) Nat.Genet. 42:790-3; Lederer et al. (2012) Am. J. Hum. Genet. 90:119-24;Miyake et al. (2013) Hum. Mutat. 34:108-10).

SUMMARY

The presently disclosed subject matter relates to the discovery that areversible deficiency of postnatal neurogenesis in the granule celllayer of the dentate gyrus associated with intellectual disability in KScan be ameliorated postnatally by agents that favor open chromatinstates. In particular, the presently disclosed subject matter providesmethods for treating KS and other Mendelian disorders of the epigeneticmachinery using agents that restore balance between open and closedchromatin states at one or more target genes.

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning. A Laboratory Manual, 3^(rd) ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. andLane, D., Antibodies—A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988; Freshney, R. I., “Culture of AnimalCells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons,Hoboken, N.J., 2005. Non-limiting information regarding therapeuticagents and human diseases is found in Goodman and Gilman's ThePharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005,Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton& Lange 10^(th) ed. (2006) or 11th edition (July 2009). Non-limitinginformation regarding genes and genetic disorders is found in McKusick,V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes andGenetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12thedition) or the more recent online database: Online MendelianInheritance in Man, OMIM™. McKusick-Nathans Institute of GeneticMedicine, Johns Hopkins University (Baltimore, Md.) and National Centerfor Biotechnology Information, National Library of Medicine (Bethesda,Md.), as of May 1, 2010, available on the World Wide Web:http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance inAnimals (OMIA), a database of genes, inherited disorders and traits inanimal species (other than human and mouse), available on the World WideWeb: http://omia.angis.org.au/contact.shtml. All patents, patentapplications, and other publications (e.g., scientific articles, books,websites, and databases) mentioned herein are incorporated by referencein their entirety. In case of a conflict between the specification andany of the incorporated references, the specification (including anyamendments thereof, which may be based on an incorporated reference),shall control. Standard art-accepted meanings of terms are used hereinunless indicated otherwise. Standard abbreviations for various terms areused herein.

The presently disclosed subject matter provides a method of treating aMendelian disorder of the epigenetic machinery in a subject in needthereof, the method comprising administering a therapeutically effectiveamount of an agent that restores balance between open and closedchromatin states at one or more target genes. In some aspects, the agentthat restores balance between open and closed chromatin states at one ormore target genes is an agent that ameliorates the effect of a defectivegene encoding a component of the epigenetic machinery. In other aspects,the subject is a postnatal human subject.

In additional aspects, the defective gene encoding a component of theepigenetic machinery encodes a histone methyltransferase. In someembodiments, when the defective gene encoding a component of theepigenetic machinery encodes a histone methyltransferase, the Mendeliandisorder of the epigenetic machinery is selected from the groupconsisting of Kabuki syndrome (KS), Wiedemann-Steiner syndrome (WSS),Kleefstra syndrome (KLFS), Weaver syndrome (WS), and Sotos syndrome(SS). In other aspects, where the defective gene encoding a component ofthe epigenetic machinery encodes a histone methyltransferase, the agentthat restores balance between open and closed chromatin states at one ormore target genes comprises a histone deacetylase inhibitor (HDACi). Infurther aspects, the HDACi is selected from the group consisting of:(S)—N-hydroxy-4-(3-methyl-2-phenylbutanamido)benzamide (AR-42);2-Propylpentanoic acid (valproic acid);N′-hydroxy-N-phenyl-octanediamide (vorinostat); and5H-dibenz[b,f]azepine-5-carboxamide (carbamazepine). In still furtheraspects, where the Mendelian disorder of the epigenetic machinery is KS,the defective gene encoding a component of the epigenetic machinery isKMT2D.

In another aspect of the presently disclosed methods, the defective geneencoding a component of the epigenetic machinery encodes a histonedeacetylase. In some embodiments, when the defective gene encoding acomponent of the epigenetic machinery encodes a histone deacetylase, theMendelian disorder of the epigenetic machinery is selected from thegroup consisting of Brachydactyly-mental retardation syndrome (BDMR),Cornelia de Lange syndrome 5 (CDLS5), and Wilson-Turner syndrome (WTS).In other aspects, where the defective gene encoding a component of theepigenetic machinery encodes a histone deacetylase, the agent thatrestores balance between open and closed chromatin states at one or moretarget genes is a histone acetyltransferase (HAT) inhibitor. In furtheraspects, the HAT inhibitor is selected from the group consisting of:(1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione(curcumin); and 2-hydroxy-6-pentadecyl-benzoic acid (anacardic acid). Instill further aspects, where the Mendelian disorder of the epigeneticmachinery is BDMR, the defective gene encoding a component of theepigenetic machinery is HDAC4.

In another aspect of the presently disclosed methods, the defective geneencoding a component of the epigenetic machinery encodes a histonedemethylase. In some embodiments, when the defective gene encoding acomponent of the epigenetic machinery encodes a histone demethylase, theMendelian disorder of the epigenetic machinery is selected from thegroup consisting of Claes-Jensen syndrome (CJS) and KS.

In another aspect of the presently disclosed methods, the defective geneencoding a component of the epigenetic machinery encodes a histoneacetyltransferase. In some embodiments, when the defective gene encodinga component of the epigenetic machinery encodes a histoneacetyltransferase, the Mendelian disorder of the epigenetic machinery isselected from the group consisting of Rubinstein-Taybi syndrome (RTS),Genitopatellar syndrome (GPS), and Say-Barber-Biesecker-Young-Simpsonsyndrome (SBBYS).

In another aspect of the presently disclosed methods, the defective geneencoding a component of the epigenetic machinery encodes a DNAmethyltransferase. In some embodiments, when the defective gene encodinga component of the epigenetic machinery encodes a DNA methyltransferase,the Mendelian disorder of the epigenetic machinery is selected from thegroup consisting of Hereditary sensory and autonomic neuropathy withdementia and hearing loss (HSAN1E), Autosomal dominant cerebellarataxia, deafness, and narcolepsy (ADCADN), and Immunodeficiency,centromeric instability, and facial anomalies syndrome (ICF).

In another aspect of the presently disclosed methods, the defective geneencoding a component of the epigenetic machinery encodes a planthomeodomain finger protein. In some embodiments, when the defective geneencoding a component of the epigenetic machinery encodes a planthomeodomain finger protein, the Mendelian disorder of the epigeneticmachinery is selected from the group consisting of Siderius X-linkedmental retardation syndrome (MRXSSD) and Borjeson-Forssman-Lehmannsyndrome (BFLS).

In another aspect of the presently disclosed methods, the defective geneencoding a component of the epigenetic machinery encodes aBromodomain-containing protein. In some embodiments, when the defectivegene encoding a component of the epigenetic machinery encodes aBromodomain-containing protein, the Mendelian disorder of the epigeneticmachinery is X-linked mental retardation and macrocephaly.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1 shows components of the epigenetic machinery. This machineryconsists of writers (highlighters) and erasers of marks (for example,trimethylation of lysine 4 on histone H3 (H3K4me3)) as well as readersof those marks. A net balance between systems that remove and add aparticular mark must be achieved;

FIG. 2 shows selected Mendelian disorders of the histone machinerycaused by alterations of writers (highlighters) and erasers. Acetylationis a binary mark (present or not), and histone lysine methylation aquaternary mark (present as mono-, di-, tri-, or unmethylated). Thediagram illustrates these two types of modifications on two of theN-terminal histone tails, histone H3 and histone H4. The writers(highlighters) and erasers place and remove the modifications; some ofthese are associated with open, permissive chromatin (green), and othersare associated with closed, repressive chromatin (red). Based on theenzymatic component of the epigenetic machinery involved and thepredicted consequence of the reported mutations for each disorder, thediagram shows conditions that would be expected to shift the balancetoward closed chromatin states at target loci (top) and conditions thatwould be expected to shift the balance toward open chromatin states attarget loci (bottom);

FIG. 3 shows therapeutic approaches based on understanding and restoringthe balance of chromatin states;

FIG. 4 shows a schematic diagram of an embodiment of the presentlydisclosed genetically encoded indicator system;

FIG. 5A and FIG. 5B show embodiments of fluorescent indicator constructsused in the presently disclosed subject matter: an acetyl-indicator(FIG. 5A); and a methyl-indicator (FIG. 5B);

FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, and FIG. 6F show that theKmt2d^(+/βGeo) mouse model of Kabuki syndrome demonstrates hippocampalmemory defects. FIG. 6A shows domain organization of MLL family members,with the relative position of the H3K4 methyltransferase SET domainindicated in red and other domains by additional colors. The human andmurine chromosomal assignment (Chr) is shown. FIG. 6B shows theKmt2d^(βGeo) targeting event introduces a β-Geo cassette including astrong splice acceptor (SA) sequence and a 3′ cleavage andpolyadenylation signal (pA) into intron 50 of Kmt2d on mouse chromosome15 (FIG. 7A). FIG. 6C shows real-time PCR using primers specific forexons 20 or 52 of Kmt2d (arrows) confirms a substantial reduction (˜50%)in mRNA corresponding to sequences distal to the β-Geo insertion sitewhen compared to proximal sequences in Kmt2d^(+/βGeo) mice, incomparison to Kmt2d^(+/+) littermates. Results reflect three technicalreplicates for each of 3 Kmt2d^(+/+) and 2 Kmt2d^(+/βGeo) mice. FIG. 6Dshows that the ChIP-seq reveals a genome-wide deficiency of H3K4me3 incells from Kmt2d^(+/βGeo) mice, when compared to cells from Kmt2d^(+/+)littermates. A positive value indicates a higher locus-specific peak inKmt2d^(+/βGeo) mice. Each point corresponds to a genomic location with apeak in at least one sample. Significantly differentially bound loci arered, while others are gray. FIG. 6E shows there was no difference inpositional preference between genotypes during the habituation phase[identical objects (L/R)]. Kmt2d^(+/βGeo) mice spent less time with anovel object placed to the left (L) of a habituated object on the right(R) compared to Kmt2d^(+/+) littermates, which also significantlyimproved from habituation phase [Novel object (L)]. n=13 (+/+), 10(+/βGeo). FIG. 6F shows that Kmt2d^(+/βGeo) mice showed a reducedfrequency in platform zone crossings during the probe trial phase ofMorris water maze testing, n=48 (+/+), 32 (+/βGeo). *P<0.05.^(†)P<0.005; ^(††)P<0.001;

FIG. 7A and FIG. 7B show the integration site of gene trap in theKmt2d^(βGeo) allele. FIG. 7A shows the DNA sequence of the targetedallele showing the sequence for Kmt2d exon 50 (red) and intron 50 (blue)and the gene trap encoding the β-Geo cassette (purple). FIG. 7B showsimmunoprecipitated protein using an antibody directed against KMT2Dshows immunoreactivity for 3-galactosidase in cellular lysates fromKmt2d^(+/βGeo) mice but not Kmt2d^(+/+) littermates. The presence ofthis hybrid protein suggests that mRNA from the Kmt2d^(βGeo) allele isboth transcribed and translated;

FIG. 8A, FIG. 8B, and FIG. 8C show that Kmt2d^(+/βGeo) mice showoverlapping phenotypic features with patients with KS: decreasedprotrusion of the maxilla over the mandible can be seen when skin isremoved (FIG. 8A) and on radiographs (FIG. 8B) in Kmt2d^(+/βGeo) mice,when compared to Kmt2d^(+/+) littermates (n>5 for both groups). FIG. 8Cshows that this was verified by a group of investigators blinded togenotype which gave Kmt2d^(+/+) mice a significantly higher maxillaryprotrusion score than Kmt2d^(+/βGeo) littermates. ^(†)P<0.005;

FIG. 9 shows that Kmt2d^(+/βGeo) mice have context related memorydefects. Kmt2d^(+/βGeo) show impaired performance in a fear conditioningassay, when compared to Kmt2d^(+/+) littermates. n=20 (+/+), 8 (+/βGeo).P<0.05 (repeated measures ANOVA comparing two genotypes in all timepoints);

FIG. 10 shows that Kmt2d^(+/βGeo) mice show no deficit in flag trial.Kmt2d^(+/βGeo) mice and Kmt2d^(+/+) littermates show similar performanceduring flag trials prior to Morris water maze testing (as reflected byno significant difference in a repeated measures ANOVA), suggesting noinherent impairment to task completion such as visual impairment, insubsequent memory-based testing. N.S., n=15 (+/+), 9 (+/βGeo);

FIG. 11A, FIG. 11B, and FIG. 11C show the assessment of motor functionin Kmt2d^(+/βGeo) and Kmt2d^(+/+) mice: Kmt2d^(+/βGeo) mice did not showany deficit in general activity level (as monitored by beam breaks inopen field testing) (FIG. 11A); or grip strength (FIG. 11B), whencompared to Kmt2d^(+/+) littermates. FIG. 11C shows comparable swimmingspeed in the probe trial of the Morris Water Maze. Open field testing:N.S., n=11 (+/+), 11 (+/βGeo); grip strength: N.S., n=18 (+/+), 8(+/βGeo). MWM probe trial: N.S., n=29 (+/+), 23 (+/βGeo);

FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D show the escape latenciesduring Morris water maze training. FIG. 12A shows average latency toplatform zone for Kmt2d^(+/βGeo) (yellow) and Kmt2d^(+/+) animals(blue). Repeated measures ANOVA showed no significant difference betweengroups across all time points. FIG. 12B show Kmt2d^(+/βGeo) mice on 10mg/kg/day of AR-42 (yellow triangle) and Kmt2d^(+/+) animals on 10mg/kg/day of AR-42 (blue circle). No significant difference is observed.FIG. 12C shows Kmt2d^(+/+) animals with (blue circle) and without (bluesquare) 10 mg/kg/day of AR-42 (significant difference with P<0.01). FIG.12D shows Kmt2d^(+/βGeo) animals with (yellow rhombus) and without(yellow triangle) 10 mg/kg/day of AR-42. No significant difference isobserved. n=32 (+/βGeo, vehicle), 44 (+/+, vehicle), 9 (+/βGeo, AR-42),14 (+/+, AR-42);

FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E, FIG. 13F, and FIG. 13Gshow that Kmt2d^(+/βGeo) mice demonstrate a global deficiency of H3K4me3in the DG associated with reduced GCL volume and neurogenesis. FIG. 13Ashows that immunofluorescence reveals intense expression of KMT2D (redsignal) in the dentate gyrus GCL and pyramidal layer of Kmt2d^(+/+)mice. FIG. 13B shows immunofluorescence showing H3K4me3 (red) and DAPI(blue) in the GCL of Kmt2d^(+/βGeo) mice and Kmt2d^(+/+) littermates.FIG. 13C shows that quantification reveals a reduced H3K4me3/DAPI signalintensity ratio within the GCL of Kmt2d^(+/βGeo) mice compared toKmt2d^(+/+) littermates. n=9 (+/+), 5 (+/βGeo). FIG. 13D shows that thecalculation of GCL area (red outline) in every sixth brain slice alloweddemonstration of reduced GCL volume. FIG. 13E shows Kmt2d^(+/βGeo) micecompared to Kmt2d^(+/+) littermates (n=4 (+/+), 5 (+/βGeo)). FIG. 13Fand FIG. 13G show that immunofluorescence reveals reduced representationof cells positive for doublecortin (DCX), a marker for neurogenesis, inthe GCL of Kmt2d^(+/βGeo) mice compared to Kmt2d^(+/+) littermates. n=4(+/+), 4 (+/βGeo). *P<0.05; ^(††)P<0.001;

FIG. 14 shows that H3K4me3 is decreased in the pyramidal layer inKmt2d^(+/βGeo) mice compared to Kmt2d^(+/+) littermates. H3K4me3 is alsosignificantly reduced in the pyramidal layer of the hippocampus, anothercell layer with strong expression of KMT2D protein. n=5 (+/+), 5(+/βGeo). **P<0.01;

FIG. 15A and FIG. 15B show the body and brain size in Kmt2d^(+/βGeo)mice. While Kmt2d^(+/βGeo) animals show a significant reduction in bodyweight, at 5 months of age when compared to Kmt2d^(+/+) littermates(FIG. 15A), there was no significant difference in brain weight (FIG.15B). Body, n=10 (+/+), 5 (+/βGeo). Brain, N.S., n=14 (+/+), 12(+/3Geo),*P<0.05;

FIG. 16 shows EdU incorporation. Kmt2d^(+/βGeo) mice showed reducedincorporation of EdU in the GCL 30 days after the onset of injection,suggesting reduced neurogenesis and long-term neuronal survival, whencompared to Kmt2d^(+/+) littermates, as assessed by observers blinded togenotype. n=7 (+/+), 4 (+/βGeo) **P<0.01;

FIG. 17 shows decreased dendrites in DCX+ cells in GCL of Kmt2d^(+/βGeo)mice. Immunofluorescence shows that Kmt2d^(+/βGeo) animals show anapparent decrease in dendritic arborization of cells that are DCX+, whencompared to Kmt2d^(+/+) littermates;

FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, FIG. 18E, FIG. 18F, FIG. 18G,FIG. 18H, and FIG. 18I show that the H3k4me3 epigenetic reporter alleledemonstrates decreased activity in Kmt2d^(+/βGeo) cells. FIG. 18A showsthe domain organization encoded by the H4ac and H3K4me3 reporteralleles. The H4ac indicator includes H4 (lysine positions indicated),the C- and N-terminal halves of E-GFP separated by a short linker (L),the TAFII binding domain (BD) and a repetitive nuclear localizationsignal (NLS). The H3K4me3 indicator includes the H3 and the TAF3-PHD.FIG. 18B shows that the recognition of the histone tail mark by therelevant histone reader leads to reconstitution of GFP structure andfunction (fluorescence). FIG. 18C and FIG. 18D show that the acetylationindicator demonstrates increasing fluorescence with increasing amountsof the histone deacetylase inhibitor SAHA. FIG. 18E shows that theactivity of the H4ac indicator is lost upon mutagenesis of all potentialacetylation sites from lysine to arginine. FIG. 18F shows that theH3K4me3 indicator demonstrates a dose dependent response to the histonedeacetylase inhibitor AR-42 with decreased cell numbers at higher doses(red line). FIG. 18G shows that the activity is greatly reduced uponmutagenesis of K4 in the H3 tail and D890A/W891A and M882A in the readerpocket. FIG. 18H shows that the H3K4me3 indicator shows reduced activityin murine embryonic fibroblasts (MEFs) derived from Kmt2d^(+/βGeo) micecompared to Kmt2d^(+/+) littermates. Both genotypes show adose-dependent response to AR-42, with Kmt2d^(+/βGeo) MEFs achievinguntreated wild-type levels of activity at a dose of 5 μM. n=3 (+/+), 3(+/βGeo), biological replicates for each dose. **P<0.01, ^(††)P<0.001.FIG. 18I shows an experiment demonstrating that lysines on H4 arerequired for activity of the acetyl reporter: a) 293 cells nottransfected but treated with 0 μM, 2.5 μM and 7.5 μM of Vorinostat (leftto right); b) 293 cells transfected with reporter that has a singleacetylation site with 0 μM, 2.5 μM and 7.5 μM of Vorinostat (left toright); c) 293 cells transfected with reporter with all possibleacetylation sites changed from lysine's to arginines with 0 μM, 2.5 μMand 7.5 μM of Vorinostat (left to right);

FIG. 19A and FIG. 19B show that SAHA increases acetylation of theindicator (FIG. 19A) and the saturation curve of the acetylationindicator as seen by % positive cells (FIG. 19B);

FIG. 20 shows that HDAC3 attenuates signal of the H4ac indicator. HEK293cells stably expressing the H4ac indicator show increased signal uponstimulation with the histone deacetylase SAHA that is attenuated byrecombinant expression of HDAC3. n=3 biologic replicates for each state,stable transfection. **P<0.01;

FIG. 21 shows that both indicators demonstrate a deficiency inKmt2d^(+/βGeo) mice. Stable expression of the specified indicator intomouse embryonic fibroblasts (MEFs) demonstrates significant deficienciesin both histone H4 acetylation and H3K4 trimethylation activity inKmt2d^(+/βGeo) MEFs compared to Kmt2d^(+/+) cells, as assessed by thepercentage of GFP positive cells. n=4 (+/+), 3 (+/βGeo). *P<0.05;

FIG. 22A and FIG. 22B show improved H3K4 trimethylation activity inKmt2d^(+/βGeo) cells transiently transfected with H3K4 trimethylationindicator and treated with MS275. FIG. 22A shows that Kmt2d^(+/βGeo)MEFs show reduced H3K4 trimethylation activity, when compared toKmt2d^(+/+) cells, that is improved upon treatment with the histonedeacetylase MS275. FIG. 22B shows that transiently transfected cells ofboth genotypes demonstrate comparable transfection efficiency asestimated by real time PCR when compared to a genomic control. n=6(+/+), 6 (+/βGeo), biological replicates for each concentration,transient transfection. *P<0.05; **P<0.01; ^(††)P<0.001;

FIG. 23 shows HDAC inhibitors with H3K4 trimethylation effects (AR-42and MS275) and an HDAC inhibitor with H3 acetylation effects (SAHA) atlow and high doses;

FIG. 24A, FIG. 24B, FIG. 24C, and FIG. 24D show the in vivo responses toAR-42. FIG. 24A and FIG. 24B show that immunofluorescence revealsincreased H3K4me3 in the GCL of Kmt2d^(+/+) and Kmt2d^(+/βGeo) mice upontreatment with 25 mg/kg/day of AR-42, with no difference betweengenotypes in the treated groups, n=4-5 per group. FIG. 24C show that 25mg/kg/day of AR-42 did not improve DCX expression in Kmt2d^(+/βGeo) miceand reduced DCX expression in Kmt2d^(+/+) animals, n=4-6 per group. FIG.24D shows that DCX expression was improved in older mice (5-6 months)upon treatment of Kmt2d^(+/βGeo) mice with 10 mg/kg/day of AR-42, n=3-4per group. *P<0.05; **P<0.01; ^(††)P<0.001;

FIG. 25A, FIG. 25B, FIG. 25C, FIG. 25D, FIG. 25E and FIG. 25F show thein vivo effects of AR-42. One to two month old mice of both genotypesshow an increase in H3K4me3 (FIG. 25A and FIG. 25B) [n=5-6 per group]associated with a dose-dependent increase in neurogenesis inKmt2d^(+/βGeo) mice (FIG. 25C and FIG. 25D) (monitored by normalized DCXexpression) [n=4-6 per group] upon treatment with the HDACi AR-42 withno difference between mutant and wild-type animals at a dose of 10mg/kg/day. FIG. 25E shows that the genome-wide deficiency of H3K4me3seen in Kmt2d^(+/βGeo) mice is improved upon treatment with 10 mg/kg/dayAR-42. FIG. 25F shows that the reduced frequency of platform crossingseen during Morris water maze testing of Kmt2d^(+/βGeo) mice wasnormalized upon treatment with 10 mg/kg/day of AR-42. [n=48 (+/+, notreatment), 32 (+/βGeo, no treatment), 14 (+/+, 10 mg/kg/day AR-42), 9(+/βGeo, 10 mg/kg/day AR-42)]. *P<0.05; **P<0.01; ^(†)P<0.005;^(††)P<0.001;

FIG. 26 shows the AR-42-induced expression of a known Kmt2d target gene.Klf10, a known target gene of Kmt2d (Guo et al. (2012) Proc. Natl. Acad.Sci. U.S.A. 109:17603-8), demonstrates reduced expression in spleencells of Kmt2d^(+/βGeo) mice that is normalized upon treatment withAR-42 n=4 per group. *P<0.05, ^(†)P<0.005;

FIG. 27A, FIG. 27B, FIG. 27C, FIG. 27D, and FIG. 27E show MA plots thatindicate a shift in the balance of H3K4me3 upon treatment with AR-42:significant differences in the locus-specific intensity of H3K4me3 areindicated in red, with the directionality and magnitude of each peakheight reflecting the difference between the indicated states (genotypeand AR-42 treatment status). Kmt2d^(+/βGeo) animals demonstrate adownward shift compared to Kmt2d^(+/+) littermates (FIG. 27A), which isrecovered with AR-42 (FIG. 27B). These data indicate there may be someovercorrection which could be improved in future studies by usingChIP-seq as a biomarker. The difference between AR-42 treated andvehicle treated Kmt2d^(+/+) animals is less notable (FIG. 27C), butobvious when comparing Kmt2d^(+/βGeo) on AR-42 compared toKmt2d^(+/βGeo) littermates on vehicle (FIG. 27D) or both genotypes onAR-42 (FIG. 27E). CPM: counts per million, FC: fold change. n=2 (+/βGeo,vehicle), 2 (+/+, vehicle), 2 (+/βGeo, AR-42), 2 (+/+, AR-42);

FIG. 28A, FIG. 28B, FIG. 28C, FIG. 28D and FIG. 28E show a visualizationof shifts in balance between states (genotype or AR-42) as a function ofintensity demonstrates an abnormality in Kmt2d^(+/βGeo) that isresponsive to AR-42: Kmt2d^(+/βGeo) animals demonstrate a downward shiftcompared to Kmt2d^(+/+) littermates (FIG. 28A; −2 log Q: 1187.1,P<2.2e−16), which is normalized (but somewhat over corrected) with AR-42(FIG. 28B; −2 log Q: 589.5, P<2.2e−16). The difference betweenAR-42-treated Kmt2d^(+/+) mice and vehicle treated Kmt2d^(+/+)littermates is less notable (FIG. 28C), but more evident when comparingKmt2d^(+/βGeo) on AR-42 to Kmt2d^(+/βGeo) littermates on vehicle (FIG.28D) or on both genotypes on AR-42 (FIG. 28E) (−2 log Q: 359.9,P<2.2e−16). n=2 (+/βGeo, vehicle), 2 (+/+, vehicle), 2 (+/βGeo, AR-42),2 (+/+, AR-42); and

FIG. 29 shows serum control experiments for antibodies used forimmunofluorescence. Non-specific binding was not observed when sectionswere sequentially exposed to serum from the same species matching theprimary antibody for each experiment (i.e. rabbit for KMT2D and H3K4me3and goat for doublecortin), followed by the secondary antibody used forKMT2D and H3K4me3 (anti-rabbit) or doublecortin (anti-goat).

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the presently disclosed subject matter areshown. Like numbers refer to like elements throughout. The presentlydisclosed subject matter may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements. Indeed, many modifications andother embodiments of the presently disclosed subject matter set forthherein will come to mind to one skilled in the art to which thepresently disclosed subject matter pertains having the benefit of theteachings presented in the foregoing descriptions and the associatedFigures. Therefore, it is to be understood that the presently disclosedsubject matter is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims.

I. Methods of Treating Mendelian Disorders of the Epigenetic Machinery

The presently disclosed subject matter provides a method of treating aMendelian disorder of the epigenetic machinery in a subject in needthereof, the method comprising administering a therapeutically effectiveamount of an agent that restores balance between open and closedchromatin states at one or more target genes is an agent thatameliorates the effect of a defective gene encoding a component of theepigenetic machinery. In particular, the presently disclosed subjectmatter relates to the discovery that a reversible deficiency ofpostnatal neurogenesis in the granule cell layer of the dentate gyrusassociated with intellectual disability in KS can be amelioratedpostnatally by agents that favor open chromatin states. The term “openchromatin states” is used herein since the agent used is an histonedeacetylase inhibitor expected to lead to an increase in histoneacetylation, a histone modification exclusively seen in open chromatin.For example, as described elsewhere herein, treatment with a histonedeacetylase inhibitor (AR-42) rescued postnatal neurogenesis in thegranule cell layer of the dentate gyrus even though the epigeneticdefect in mouse model of KS has to do with histone methylation system (adeficiency of H3K4me3 mark, see FIG. 6D). H3K4me3, like histoneacetylation, is an open chromatin mark. Therefore, these results showedthat one epigenetic modification can be targeted (histone acetylation)to make up for a deficiency in another epigenetic modification (histonemethylation). In addition, these results showed that balancing thechromatin states of target genes, either by favoring open chromatin indiseases with a defect in epigenetic component leading to increasedclosed chromatin (see KS in FIG. 3) or favoring closed chromatin indisease with too much open chromatin (see BDMR in FIG. 3) offers ageneralized treatment strategy for this group of disorders—the Mendeliandisorders of the histone machinery.

Balance Between Open and Closed Chromatin States

The DNA methylation machinery and the histone machinery affect theexpression of many genes in trans (Berdasco & Esteller (2013) Hum.Genet. 132:359-83; Wolffe (1994) Trends Biochem. Sci. 19:240-44). Withinthis group, genetic mutations may occur in writers, erasers, or readersof epigenetic marks. The writers of epigenetic marks, which can beconceptualized as a set of highlighters, place the appropriatemodifications on particular regions of the genome based on the celltype, developmental stage, and metabolic state of the cell. These marks“highlight” individual regions for use or disuse depending on whetherthe mark favors a more open or more closed chromatin state (FIG. 1). Theerasers of epigenetic marks remove these same marks, favoring theopposite chromatin states (FIG. 1). The readers of epigenetic marksrecognize and interpret particular marks locally and give cells amechanism for keeping track of the overall chromatin state (FIG. 1).

Components of the epigenetic machinery are shown in FIG. 1. Thismachinery consists of writers (highlighters) and erasers of marks (forexample, trimethylation of lysine 4 on histone H3 (H3K4me3)) as well asreaders of those marks. A net balance between systems that remove andadd a particular mark must be achieved. In many ways, the interactingepigenetic systems have certain distinct aspects that make them powerfulfinal integrators of cellular signals (Jaenisch and Bird (2003) Nat.Genet. 33(Suppl.):245-545). For instance, many of the marksplaced/removed by writers/erasers can directly affect gene expression,either in a permissive (H3K4me3, shown) or nonpermissive (H3K9me3, notshown) manner. This change in expression, presumably of multiple genes,has the potential to form feedback loops by affecting the amount andavailability of the modification in question. Various internalmetabolites can directly affect the prevalence of marks. For instance,S-adenosyl-methionine (SAM) is a donor for methylation reactions,including both DNA and histone methylation. Use of critical metabolicintermediates like SAM as donors for histone tail modifications or forDNA methylation allows environmental influences to impact and beintegrated into the system and to potentially affect gene expressiondirectly (Lu and Thompson (2012) Cell Metab. 16:9-177).

To ensure appropriate cell type-specific gene expression, a balance mustbe achieved between the activity of the two opposing systems (writersand erasers) and the subsequent placement of their respective marks(FIG. 1), ensuring that the appropriate composition of chromatin ispresent at particular gene promoters. Although a steady-state balance ofchromatin marks is likely achieved at any given time, the opposinghistone systems are likely to be dynamic in nature (Ficz et al. (2005)Development 132:3963-76; Mito et al. (2007) Science 315:1408-11),allowing the cell to rapidly respond to changes in environmental signalsby altering gene expression at specific loci. The histone machinery (aswell as some components of the DNA methylation machinery) is enormouslyredundant, perhaps reflecting the critical importance of maintainingthis balance in many different cell types (FIG. 1).

Multiple lines of evidence suggest that Mendelian disorders of theepigenetic machinery result from perturbations of a delicate balancebetween open and closed chromatin states at one or more target genes isan agent that ameliorates the effect of a defective gene encoding acomponent of the epigenetic machinery. Many of these conditions canresult from both point mutations within a gene and chromosomemicrodeletions containing the same gene, suggesting haploinsufficiencyas a common disease mechanism (e.g., Breuning et al. (1993) Am. J. Hum.Genet. 52:249-54; Kleefstra et al. (2006) Am. J. Hum. Genet. 79:370-77;Kurotaki et al. (2002) Nat. Genet. 30:365-66; Lederer et al. (2012) Am.J. Hum. Genet. 90:119-24). The majority of the writer/eraser systems,which are composed of enzymes that either add or remove chromatinmodifications, demonstrate autosomal dominant inheritance (see Table 1).This is intriguing because, in contrast, the majority of other Mendelianenzyme deficiencies are autosomal recessive conditions. Given theprolific efficacy of most enzymes, biallelic mutations with concomitantreduction of enzymatic activity to <5% of control levels are necessaryto cause the expression of disease phenotypes; heterozygotes retainingapproximately 50% of enzymatic activity are typically asymptomatic.

TABLE 1 Mendelian disorders of the epigenetic machinery. EpigeneticInheritance function Gene Gene function Disease pattern Disorders of theDNA methylation machinery Writer DNMT1 DNA methyltransferase Hereditarysensory and AD autonomic neuropathy with dementia and hearing loss(HSAN1E) Autosomal dominant AD cerebellar ataxia, deafness, andnarcolepsy (ADCADN) DNMT3b DNA methyltransferase Immunodeficiency, ARcentromeric instability, and facial anomalies syndrome (ICF) ReaderMeCP2 Methyl-CpG binding Rett syndrome XL protein MBD5 Methyl-CpGbinding 2q23.1 microdeletion/ AD protein microduplication syndromeDisorders of the histone machinery Writer CREBBP Histoneacetyltransferase Rubinstein-Taybi AD syndrome (RTS) EP300 Histoneacetyltransferase Rubinstein-Taybi AD syndrome (RTS) KAT6B Histoneacetyltransferase Genitopatellar syndrome AR (GPS) Say-Barber-Biesecker-AD Young-Simpson syndrome (SBBYS) KMT2D Histone methyltransferase Kabukisyndrome (KS) AD (or MLL2) (H3K4) MLL Histone methyltransferaseWiedemann-Steiner AD (H3K4) syndrome (WSS) EHMT1 Histonemethyltransferase Kleefstra syndrome AD (H3K9) (KLFS) EZH2 Histonemethyltransferase Weaver syndrome (WS) AD (H3K27) NSD1 Histonemethyltransferase Sotos syndrome (SS) AD (H3K36, H4K20) Eraser HDAC4Histone deacetylase Brachydactyly-mental AD retardation syndrome (BDMR)HDAC8 Histone deacetylase Cornelia de Lange XL syndrome 5 (CDLS5)Wilson-Turner syndrome XL (WTS) KDM5C* Histone demethylase Claes-Jensensyndrome XL (H3K4) (CJS) KDM6A* Histone demethylase Kabuki syndrome (KS)XL (H3K27) PHF8** Plant homeodomain Siderius X-linked mental XL fingerprotein retardation syndrome (MRXSSD) Reader PHF6 Plant homeodomainBorjeson-Forssman- XL finger protein Lehmann syndrome (BFLS) BRWD3Bromodomain-containing X-linked mental XL protein retardation andmacrocephaly *Indicates genes known to escape X-inactivation.**Indicates gene encoding a component of the epigenetic machinery thatfunctions as both an eraser and a reader. Abbreviations: AD, autosomaldominant; AR, autosomal recessive; XL, X linked.

The dosage sensitivity for Mendelian disorders of the epigeneticmachinery is intriguing in light of the enormous redundancy of some ofthe protein components with apparently overlapping functions. Forinstance, there are dozens of enzymes described with H3K4me3 activity,many of which are ubiquitously expressed (Eissenberg & Shilatifard(2010) Dev. Biol. 339:240-49). Despite this seemingly evidentredundancy, loss of a single allele of one of these enzymes issufficient to cause the multisystem disease KS. However, without beingbound by theory, perhaps this redundancy limits the number of affectedcell-types and thereby leads to a more benign phenotype that iscompatible with life despite the wide expression of this component ofthe epigenetic machinery. Although alternative explanations exist fordosage sensitivity in humans, and without being bound by theory, onehypothesis is that the total activity of writers and erasers, theepigenetic marks that they place, and thus the resulting open (active)and closed (silent) chromatin states, must remain tightly balanced incells at particular gene promoters (FIG. 1). The dosage sensitivity ofcomponents of the epigenetic machinery could suggest that levels ofcoactivators are carefully controlled in cells to fine-tune geneexpression levels. For instance, cellular systems with CREBBP deficiencyreveal dose-dependent effects on gene expression, which are dependent onthe diversity of modification systems available for a particular targetsite and/or the strength of recruitment to a particular site (Kasper etal. (2010) EMBO J. 29:3660-72). Alternatively, given that eachindividual epigenetic player within a cell acts on multiple downstreamtarget genes and genomic regions and that each works in closecooperation with transcription factors, which are also known to befinely titrated, disruption of just one allele of one of these genes issufficient to alter this delicate balance.

A balance hypothesis is supported by the observation that KS exhibitsidentical phenotypes upon disruption of either a writer of H3K4me3 (anactive mark) or an eraser of H3K27me3 (a silencing mark). This indicatesthat the transition between open and closed chromatin states is criticalto disease pathogenesis. Similarly, defects in either deposition ofhistone acetylation (RTS) or removal of histone acetylation marks (BDMR)lead to disease phenotypes. A one-sided deficiency might be expected tohave genome-wide effects, but perhaps similarly to ATRX, there are alimited number of disease-relevant target genes that are particularlysensitive in each case. At target gene promoters in cellshaploinsufficient for MLL2, for example, one might expect to seedecreased H3K4me3 and thus increased H3K4me0. Although most genes mighttolerate this deficiency, perhaps a small number of disease-relevanttarget genes in key cell populations are critically affected by thisdisrupted balance, resulting in altered gene expression. Alternatively,this might promote placement of other marks—possibly H3K9me3, DNAmethylation, and H3K27me3—which also could affect transcription.

A number of Mendelian disorders of the epigenetic machinery are known toinvolve disruption of histone writers and erasers (See Table 1). If thedelicate balance of histone marks and the subsequent effects on thepredisposition toward open or closed chromatin states are central topathogenesis, then it might be critical to determine whether the effectof disruption of each component of the epigenetic machinery would beexpected to lead to a defect in open chromatin (likely leading todownregulation of gene expression) or a defect of closed chromatin(likely leading to upregulation of gene expression) at downstream targetgenes. FIG. 2 categorizes disorders based on expected effects onchromatin states.

Furthermore, the hypothesis that a delicate balance of chromatin marksand target gene expression is important in the pathogenesis of Mendeliandisorders of the epigenetic machinery is not limited to the histonemodification system. Disruption of a dosage-sensitive reader ofepigenetic marks, MBD5, can lead to similar phenotypic differences ifMBD5 is deleted or duplicated. Indeed, MBD5 expression is reduced inindividuals with haploinsufficiency of the predicted reader ofepigenetic marks, regardless of mutation mechanism (Talkowski et al.(2011) Am. J. Hum. Genet. 89:551-63), and MBD5 expression is increasedwhen the region is duplicated (Mullegama et al. (2014) Eur. J. Hum.Genet. 22:57-63). However, downstream epigenetic and cellularconsequences and particular target loci have not been identified.Similarly, a protein closely related to MBD5 and a known reader of DNAcytosine methylation, MeCP2, exhibits marked dosage sensitivity, whichis illustrated nicely by the varying phenotypic severity of thecorresponding X-linked disorder, Rett syndrome, based on the number ofcopies of the MeCP2 gene expressed (Guy et al. (2011) Annu. Rev. CellDev. Biol. 27:631-52). The disorder is more severe in males, who have nofunctional copies of the gene; for females, the phenotype can varygreatly depending on the pattern of X inactivation, as well as on thetype and severity of mutation. The more skewed the X inactivationpattern is toward the normal X, the less severe the disease phenotype(Guy et al. (2011) Annu. Rev. Cell Dev. Biol. 27:631-52). That bothMeCP2 and MBD5, two readers of epigenetic marks, appear to be soexquisitely dosage sensitive suggests the importance of maintaining notonly a critical balance of epigenetic marks and chromatin states withincells but also a critical number of interpreters of those marks.Furthermore, the phenotypic findings in these disorders along with thedistinct neurobehavioral-predominant pathogenic features of each suggestthat this balance is particularly delicate within the central nervoussystem.

It was hypothesized that observed gene dosage sensitivity in KS involveda relative imbalance between open and closed chromatin states forcritical target genes. If this was the case, it was hypothesized thatthis balance could be restored with drugs that promote open chromatinstates, such as a histone deacetylase inhibitor (HDACi). The presentlydisclosed subject matter relates to the discovery that a reversibledeficiency of postnatal neurogenesis in the granule cell layer of thedentate gyrus associated with intellectual disability in KS can beameliorated postnatally by agents that favor open chromatin states, asdescribed more fully in the Examples below.

A. Mendelian Disorders of the Epigenetic Machinery

Selected Mendelian disorders of the histone machinery caused byalterations of writers (highlighters) and erasers are shown in FIG. 2.Acetylation is a binary mark (present or not), and histone lysinemethylation a quaternary mark (present as mono-, di-, tri-, orunmethylated). The diagram illustrates these two types of modificationson two of the N-terminal histone tails, histone H3 and histone H4. Thewriters (highlighters) and erasers place and remove the modifications;some of these are associated with open, permissive chromatin (FIG. 2,green), and others are associated with closed, repressive chromatin(FIG. 2, red). Based on the enzymatic component of the epigeneticmachinery involved and the predicted consequence of the reportedmutations for each disorder, the diagram shows conditions that would beexpected to shift the balance toward closed chromatin states at targetloci (FIG. 2, top) and conditions that would be expected to shift thebalance toward open chromatin states at target loci (FIG. 2, bottom).The former category includes Rubinstein-Taybi syndrome (RTS) (Petrij etal. (1995) Nature 376:348-5192; Roelfsema et al. (2005) Am. J. Hum.Genet. 76:572-80), Kabuki syndrome (KS) (Lederer et al. (2012) Am. J.Hum. Genet. 90:119-24; Ng et al. (2010) Nat. Genet. 42:790-93),Wiedemann-Steiner syndrome (WSS) (Jones et al. (2012) Am. J. Hum. Genet.91:358-64), and possibly Weaver syndrome (WS) and Sotos syndrome (SS)(Gibson et al. (2012) Am. J. Hum. Genet. 90:110-18; Tatton-Brown et al.(2011) Oncotarget 2:1127-33; Kurotaki et al. (2002) Nat. Genet.30:365-66); the latter category includes brachydactyly-mentalretardation syndrome (BDMR) (Williams et al. (2010) Am. J. Hum. Genet.87:219-28), Kleefstra syndrome (KLFS) (Kleefstra et al. (2006) Am. J.Hum. Genet. 79:370-776), Claes-Jensen syndrome (CJS) (Jensen et al.(2005). Am. J. Hum. Genet. 76:227-366), and Sotos syndrome (SS)(Kurotaki et al. (2002) Nat. Genet. 30:365-666).

Accordingly, in some embodiments of the methods of the presentlydisclosed subject matter for treating a Mendelian disorder of theepigenetic machinery in a subject in need thereof, the defective geneencoding a component of the epigenetic machinery encodes a histonemethyltransferase and the Mendelian disorder of the epigenetic machineryis selected from the group consisting of Kabuki syndrome (KS),Wiedemann-Steiner syndrome (WSS), Kleefstra syndrome (KLFS), Weaversyndrome (WS), and Sotos syndrome (SS). In still further embodiments,where the Mendelian disorder of the epigenetic machinery is KS, thedefective gene encoding a component of the epigenetic machinery isKMT2D.

KS, which can be caused by a defect in either a writer or an eraser(FIG. 2). KS is an autosomal dominant or X-linked intellectualdisability syndrome with specific dysmorphic features, including aflattened facial appearance with characteristic eyes exhibiting longpalpebral fissures, eversion of the lower lids, highly arched eyebrows,and long eyelashes, as well as short stature.

KS is caused by heterozygous loss-of-function mutations in either of twogenes with complementary functions, lysine-specific methyltransferase 2D(KMT2D) on human chromosome 12 (also known as mixed lineage leukemia 2or MLL2; Ng et al. (2010) Nat. Genet. 42:790-3) or lysine-specificdemethylase 6A (KDM6A) on human chromosome X (Lederer et al. (2012) Am.J. Hum. Genet. 90:119-24) (FIG. 2). KMT2D is a methyltransferase thatadds a trimethylation mark to H3K4 (H3K4me3, an open chromatin mark)while KDM6A is a demethylase that removes trimethylation from histone 3lysine 27 (H3K27me3, a closed chromatin mark). Both genes facilitate theopening of chromatin and promote gene expression (Ng et al. (2010) Nat.Genet. 42:790-3; Lederer et al. (2012) Am. J. Hum. Genet. 90:119-24;Miyake et al. (2013) Hum. Mutat. 34:108-10). These defects lead toindistinguishable conditions (KS1 and KS2).

It may seem counterintuitive that loss-of-function mutations in a writerand an eraser lead to similar phenotypes; however, this makes sense whenthe specific functions are examined. KDM6A is a histone H3K4methyltransferase that adds trimethylation to H3K4 (Issaeva et al.(2006) Mol. Cell. Biol. 27:1889-903), a mark exclusively seen in openchromatin (ENCODE Proj. Consort. (2007) Nature 447:799-816). KDM6A is ademethylase that removes trimethylation from H3K27, a closed chromatinmark (ENCODE Proj. Consort. (2007) Nature 447:799-816; Sengoku &Yokoyama (2011) Genes Dev. 25:2266-77). KDM6A is a transcriptionalcoactivator that interacts with transcriptional machinery at thepromoters of target genes to facilitate gene expression (Issaeva et al.(2006) Mol. Cell. Biol. 27:1889-903). Decreased KDM6A (or KDM6A) levelswould therefore be expected to interfere with the upregulation ofnumerous critical target genes in susceptible cell types. Interestingly,KDM6A is a Trithorax ortholog, but in Drosophila the opposingTrithorax/Polycomb systems are known to compete to establish a dynamicbalance that determines the gene expression levels of particular targetgenes (Schwartz & Pirrotta (2008) Curr. Opin. Cell Biol. 20:266-73).

A novel mouse model of KS with deletion of the SET domain of KDM6A(KMT2D) was recently characterized that appears to have a significantdeficiency of H3K4me3 in the granule cell layer of the dentate gyrus,which is associated with defective neurogenesis and hippocampal memorydefects (see Experimental Section below). This work indicates thatspecific cell populations may have particular sensitivity to decreasedlevels of histone-modifying enzymes and the marks that they place. Thefact that a deficiency in either removing a closed chromatin mark orplacing an open chromatin mark leads to the same disease state suggeststhat the balance between systems that place open and closed chromatinmarks at particular loci may be central to the pathogenesis of KS.

In another embodiment of the presently disclosed methods, the defectivegene encoding a component of the epigenetic machinery encodes a histonedeacetylase and the Mendelian disorder of the epigenetic machinery isselected from the group consisting of Brachydactyly-mental retardationsyndrome (BDMR), Cornelia de Lange syndrome 5 (CDLS5), and Wilson-Turnersyndrome (WTS). In still further embodiments, where the Mendeliandisorder of the epigenetic machinery is BDMR, the defective geneencoding a component of the epigenetic machinery is HDAC4.

Brachydactyly-mental retardation syndrome (BDMR) provides an example ofthe opposite scenario: a disorder whose molecular abnormality tips thebalance toward open chromatin at target loci (Williams et al. (2010) Am.J. Hum. Genet. 87:219-2) (FIG. 2). In this condition, caused byhaploinsufficiency of a histone deacetylase gene (HDAC4), patients haveskeletal abnormalities, including brachycephaly and brachydactyly, aswell as intellectual disability (Williams et al. (2010) Am. J. Hum.Genet. 87:219-2). This condition also demonstrates dosage sensitivity,as the severity of the phenotype appears to be dictated by the amount ofresidual function of HDAC4 (Morris et al. (2012) Am. J. Med. Genet. A158A:2015-20). HDAC4 is an eraser of the same marks deposited byCREBBP/EP300 (Wang et al. (1999) Mol. Cell. Biol. 19:7816-27), tiltingthe balance toward closed chromatin states. For instance, decreasedamounts of HDAC4 appear to upregulate the MEF2 gene in neurons, apossible explanation for the intellectual disability seen in thesyndrome (Ronan et al. (2013) Nat. Rev. Genet. 14:347-59). In addition,HDAC4^(−/−) mice have skeletal abnormalities resembling those of humanswith BDMR, as well as increased expression of RUNX2, a known target ofHDAC4 repression (Vega et al. (2004) Cell 119:555-66). Therefore, eithertoo much or too little open chromatin at target genes can lead todisease, indicating that the balance between open and closed chromatinstates at those sites must be tightly regulated.

In another embodiment of the presently disclosed methods, the defectivegene encoding a component of the epigenetic machinery encodes a histoneacetyltransferase and the Mendelian disorder of the epigenetic machineryis selected from the group consisting of Rubinstein-Taybi syndrome(RTS), Genitopatellar syndrome (GPS), andSay-Barber-Biesecker-Young-Simpson syndrome (SBBYS).

Rubinstein-Taybi syndrome (RTS), is inherited in an autosomal dominantmanner and is characterized by specific dysmorphic features, includingtalon cusps, broad thumbs and great toes with angulation, a grimacingsmile, short stature, and intellectual disability (Petrij et al. (1995)Nature 376:348-51; Roelfsema et al. (2005) Am. J. Hum. Genet.76:572-80). The condition is caused by haploinsufficiency of either oftwo histone acetyltransferase enzyme genes (CREBBP and EP300) (Petrij etal. (1995) Nature 376:348-51; Roelfsema et al. (2005) Am. J. Hum. Genet.76:572-80), which have overlapping functions (Bannister & Kouzarides(1996) Nature 384:641-43; Ogryzko et al. (1996) Cell 87:953-59) (FIG.2). RTS is therefore a deficiency of two different writers, bothtargeting critical sites on histone tails and leading to the samephenotype. Histone acetylation is a binary mark (present or not) and isseen exclusively in open chromatin. The fact that a defect in eitherCREBBP or EP300 leads to highly overlapping phenotypes might indicatethat both histone acetyltransferases are targeted to an overlapping setof genes. Lymphoblastic cell lines from patients with mutations inCREBBP demonstrate global deficiency of histone acetylation(Lopez-Atalaya et al. (2012) J. Med. Genet. 49:66-74), and mouse modelswith targeted CREBBP alleles have demonstrated hippocampal memorydefects associated with reduced histone acetylation (Alarcón et al.(2004) Neuron 42:947-59; Korzus et al. (2004) Neuron 42:961-72; Valor etal. (2011) J. Neurosci. 31:1652-63). A deficiency of histone acetylationmight lead to a deficiency of open chromatin states in critical cellpopulations, with correspondingly lowered levels of target geneexpression. Although both KS and RTS might be expected to tilt thebalance toward less open chromatin states, the phenotypes are quitedifferent. This probably reflects different target gene specificitiesfor the KDM6A/KDM6A (Guo et al. (2012) Proc. Natl. Acad. Sci. USA109:17603-8) and CREBBP/EP300 (Wood et al. (2006) Learn. Mem. 13:609-17)systems.

In another embodiment of the presently disclosed methods, the defectivegene encoding a component of the epigenetic machinery encodes a planthomeodomain finger protein and the Mendelian disorder of the epigeneticmachinery is selected from the group consisting of Siderius X-linkedmental retardation syndrome (MRXSSD) and Borjeson-Forssman-Lehmannsyndrome (BFLS).

Although the deposition of epigenetic modifications can directly affectexpression, reading of those marks must also be important to coordinatemembers of the epigenetic machinery (Portela & Esteller (2010) Nat.Biotechnol. 28:1057-68), link chromatin-modifying systems to othersystems (Yun et al. (2011) Cell Res. 21:564-78), and ultimately affectdownstream gene expression. In that regard, some readers containdistinct domains that confer the ability to place histone modifications(writers) or remodel chromatin (Qiu et al. (2010) Cell Res. 20:908-18).Borjeson-Forssman-Lehmann syndrome (BFLS), however, is an X-linkedrecessive intellectual disability syndrome caused by missense mutationsin PHF6, encoding a protein that appears to be a reader without otherfunctional domains (Lower et al. (2002) Nat. Genet. 32:661-65), althoughPHF6 is known to bind to the NuRD complex (Denslow & Wade (2007)Oncogene 26:5433-38; Todd & Picketts (2012) J. Proteome Res. 11:4326-37)and may help to lock in repression through this interaction. BFLSpatients have intellectual disability, dysmorphic features, and obesity(Turner et al. (1989) Am. J. Med. Genet. 34:463-69). Loss-of-functionmutations in the same gene have also been found in a number of leukemias(Todd & Picketts (2012) J. Proteome Res. 11:4326-37).

In another embodiment of the presently disclosed methods, the defectivegene encoding a component of the epigenetic machinery encodes a histonedemethylase and the Mendelian disorder of the epigenetic machinery isselected from the group consisting of Claes-Jensen syndrome (CJS) andKS.

In another embodiment of the presently disclosed methods, the defectivegene encoding a component of the epigenetic machinery encodes a DNAmethyltransferase and the Mendelian disorder of the epigenetic machineryis selected from the group consisting of Hereditary sensory andautonomic neuropathy with dementia and hearing loss (HSAN1E), Autosomaldominant cerebellar ataxia, deafness, and narcolepsy (ADCADN), andImmunodeficiency, centromeric instability, and facial anomalies syndrome(ICF).

In another embodiment of the presently disclosed methods, the defectivegene encoding a component of the epigenetic machinery encodes aBromodomain-containing protein and the Mendelian disorder of theepigenetic machinery is X-linked mental retardation and macrocephaly.

B. Agents That Restore Balance Between Open and Closed Chromatin States

Therapeutic approaches based on understanding and restoring the balanceof chromatin states are shown in FIG. 3. If abnormalities of theexpression of target genes are the culprit, the target genes would beexpected to be fully functional, albeit improperly expressed, inpatients with these disorders. For instance, Kabuki syndrome (KS) isrelated to a deficiency of trimethylation of lysine 4 on histone H3(H3K4me3) or an inability to remove H3K27me3, marks that arepredominantly seen in open and repressive chromatin, respectively. Ifthe pathophysiology of KS is related to an imbalance between open andclosed chromatin states (FIG. 3, top left), with an inability to usecritical gene transcripts, then this balance could be restored byinhibiting the transition to closed chromatin with a histone deacetylase(HDAC) inhibitor (FIG. 3, bottom left). In contrast,brachydactyly-mental retardation syndrome (BDMR) would be expected tolead to an overrepresentation of open chromatin states (FIG. 3, topright), with excessive transcription of disease-relevant target genes.Therefore, a histone acetyltransferase (HAT) inhibitor could be a usefultherapeutic strategy (FIG. 3, bottom right).

Accordingly, in some embodiments of the methods of the presentlydisclosed subject matter for treating a Mendelian disorder of theepigenetic machinery in a subject in need thereof, the defective geneencoding a component of the epigenetic machinery encodes a histonemethyltransferase and the agent that restores balance between open andclosed chromatin states at the defective gene encoding a component ofthe epigenetic machinery is a histone deacetylase inhibitor (HDACi).

HDACi compounds induce hyperacetylation of histones that modulatechromatin structure and gene expression. These inhibitors also inducegrowth arrest, cell differentiation, and apoptosis of tumor cells.Recently it was reported that HDACi can restore the expression offunctional ERα to ER-breast cancer cells (Sharma et al. (2006) CancerRes. 66:6370-8; Yang et al. (2000) Cancer Res. 60:6890-4; Keen et al.(2003) Breast Cancer Res. Treat. 81:177-86). The discovery ofrecruitment of histone deacetylase (HDAC) enzymes in cancer has provideda rationale for using inhibition of HDAC activity to releasetranscriptional repression as viable option toward achieving eventualtherapeutic benefit (Vigushin et al. (2002) Anticancer Drugs 13:1-13).HDACi compounds block deacetylation function, causing cell cycle arrest,differentiation, and/or apoptosis of many tumors (Vigushin et al. (2002)Anticancer Drugs 13:1-13). Silencing of genes that affect growth anddifferentiation has been shown to occur by aberrant DNA methylation inpromoter region and by changes in chromatin structure that involvehistone deacetylation. Recent studies have established a link betweenoncogene-mediated suppression of transcription and recruitment of HDACinto nuclear complex. HDACi compounds such as butyric acid (BA),4-phenylbutyric acid and trichostatin A reverse this suppression byspecific inhibition of HDAC activity, leading to histonehyperacetylation, chromatin relaxation, and enhanced transcription.

The HDACs are a family including at least eighteen enzymes, grouped inthree classes (Class I, II and III). Class I HDACs include, but are notlimited to, HDACs 1, 2, 3, 8 and 11. Class I HDACs can be found in thenucleus and are believed to be involved with transcriptional controlrepressors. Class II HDACs include, but are not limited to, HDACs 4, 5,6, 7, and 9 and can be found in both the cytoplasm as well as thenucleus. Class III HDACs are believed to be NAD dependent proteins andinclude, but are not limited to, members of the Sirtuin family ofproteins. Non-limiting examples of sirtuin proteins include SIRT1-7. Asused herein, the term “selective HDAC” refers to an HDAC inhibitor thatdoes not substantially interact with all three HDAC classes. The term“Class I Selective HDAC” refers to an HDAC inhibitor that does notsubstantially interact with Class ii or Class II HDACs.

In one embodiment, the HDACi is selected from the group consisting of:(S)—N-hydroxy-4-(3-methyl-2-phenylbutanamido)benzamide (AR-42);2-Propylpentanoic acid (valproic acid);N′-hydroxy-N-phenyl-octanediamide (vorinostat); and5H-dibenz[b,f]azepine-5-carboxamide (carbamazepine).

In various embodiments, the HDAC inhibitor is a non-selective HDACinhibitor. In specific embodiments, the non-selective HDAC inhibitorincludes, but is not limited to:

-   (S)—N-hydroxy-4-(3-methyl-2-phenylbutanamido)benzamide (AR-42).-   2-Propylpentanoic acid (valproic acid);-   N′-hydroxy-N-phenyl-octanediamide (suberoylanilide hydroxamic acid    or SAHA or vorinostat);-   5H-dibenz[b,f]azepine-5-carboxamide (carbamazepine);-   N-Hydroxy-N′-3-pyridinyloctanediamide (pyroxamide);-   N-hydroxy-3-[3-(hydroxyamino)-3-oxo-1-propen-1-yl]-benzamide (CBHA);-   7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide    (trichostatin A or TSA);-   7-[4-(Dimethylamino)phenyl]-N—(β-D-glucopyranosyloxy)-4,6-dimethyl-7-oxo-2,4-heptadienamide    (trichostatin C);-   2-Hydroxybenzenecarbohydroxamic acid (salicylihydroxamic acid or    SBHA);-   N,N′-dihydroxynonanediamide (azelaic bihydroxamic acid or ABHA);-   azelaic-1-hydroxamate-9-anilide (AAHA);-   (1S,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetrazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone    (romidepsin or FK228);-   6-(3-chlorophenylureido) carpoic hydroxamic acid (3Cl-UCHA);-   (2E)-5-[3-(Phenylsulfonylamino)phenyl]-pent-2-en-4-ynohydroxamic    acid (oxamflatin);-   7-[4-(4-cyanophenyl)phenoxy]-heptanohydroxamic acid (A-161906);-   6-(1,3-Dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid    hydroxyamide (scriptaid);-   N-hydroxy-3-[3-](phylamino)sulfonyl phenyl]-2-propenamide    (Belinostat or PXD-101);-   (E)-3-(4-(((2-(1H-indol-3-yl)ethyl)(2-hydroxyethyl)amino)methyl)phenyl)-N-hydroxyacrylamide    (LAQ-824);-   MW2796 (see Andrews et al. (2000) International J. Parasitology 30,    761-768);-   (2E)-N-hydroxy-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]acrylamide    (Panobinostat or LBH589);-   4-acetylamino-N-(2′-aminophenyl)-benzamide (CI-994);-   N-hydroxy-2-(4-(naphthalen-2-ylsulfonyl)piperazin-1-yl)pyrimidine-5-carboxamide    (R306465);-   {6-[(diethylamino)methyl]naphthalen-2-yl}methyl    [4-(hydroxycarbamoyl)phenyl]carbamate (givinostat or ITF2357);-   3-[(Dimethylamino)methyl]-N-{2-[4-(hydroxycarbamoyl)phenoxy]ethyl}-1-benzofuran-2-carboxamide    (abexinostat or PCI-24781);-   3-[2-butyl-1-[2-(diethylamino)ethyl]-1H-benzimidazol-5-yl]-N-hydroxy-2E-propenamide    (SB-939);-   MW2996 (see Andrews et al. (2000) International J. Parasitology 30,    761-768);-   (E)-3-(1-((4-((dimethylamino)methyl)phenyl)sulfonyl)-1H-pyrrol-3-yl)-N-hydroxyacrylamide    (resminostat); or-   7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide    (CUDC-101).

In other embodiments, the HDACi is a Class I Selective HDACi, includingbut not limited to:

-   N-(2-amino-phenyl)-4-[(4-pyridin-3-yl-pyrimidin-2-ylamino)-methyl]-benzamide)    (MGCD-0103 or mocetinostat);-   Pyridin-3-ylmethyl    N-[[4-[(2-aminophenyl)carbamoyl]phenyl]methyl]carbamate (Entinostat    or SNDX-275 or MS-275);-   (1S,5S,6R,9S,15E,20R)-5-hydroxy-20-methyl-6-(propan-2-yl)-2-oxa-11,12,dithia-7,19,22-triazabicyclo[7.7.6]docos-15-ene-3,8,18,21-tetrone    (spiruchostatin A);-   4-(dimethylamino)-N-[[4-[(E)-3-(hydroxyamino)-3-oxoprop-1-enyl]phenyl]methyl]benzamide    (SK-7041);-   SK7068 (see Park et al. (2002) Eur. J. Cancer 38: Abst 318); or-   6-amino nicotinamides.

In further embodiments, the HDACi is selected from one of the followinggroups: short-chain fatty acids; hydroxamic acids;epoxyketone-containing cyclic tetrapeptides; non-epoxyketone-containingcyclic tetrapeptides; benzamides; and other miscellaneous structures(e.g., Savicol, Bacecca, MG98, Depudecin, Organosulfur compounds).Short-chain fatty acids include butyrate and phenylbutyrate,isovalerate, valproate, 4-phenyl butyrate (4-PBA), phenylbutyratepropionate, butyaramide, isobutyaramide, phenylacetate,3-bromopropionate, tributyrin, valproic acid, and Pivanex. Hydroxamicacids include the trichostatins such as TSA and TSC, SAHA and itsderivatives, Oxamflatin, ABHA, AAHA, SBHA, CBHA, pyrozamide,salicylbishyudoxamic acid, Scriptaid, Pyroxamide, Propenamides, LBH589,CHAP, MY29996, MW2976, and any of the hydroximic acids disclosed in U.S.Pat. Nos. 5,369,108; 5,932,616; 5,700,811; 6,087,367; and 6,511,990.Epoxyketone-containing cyclic tetrapeptides include trapoxins,depeudecin, depsipeptide FK228, FR 225497, Apicidin, cyclictetrapeptide, Apicidin Ia, Apicidin Ib, Apicidin Ic, Apicidin IIIa,Apicidin III, a cyclic tetrapeptide containing a2-amino-8-oxo-9,10-epoxy-decanoyl moitey, a cyclic peptide without the2-amino-8-oxo-9, 10 epoxy-decanoyl moity, HC-toxin, Chlamydocin,Diheteropeptin, WF-3161, Cyl-1 and Cyl-2). Non-epoxyketone-containingcyclic tetrapeptides include FR901228, Apicidin,cyclic-hydroxamic-acid-containing peptides (CHAPs). Benzamides includeMS-275, N-acetyldinaline, CI-994, MGCD0103, AR-42, and other benzamideanalogs.

In other embodiments of the methods of the presently disclosed subjectmatter for treating a Mendelian disorder of the epigenetic machinery ina subject in need thereof, the defective gene encoding a component ofthe epigenetic machinery encodes a histone deacetylase, and the agentthat restores balance between open and closed chromatin states at one ormore target genes is a histone acetyltransferase (HAT) inhibitor. Infurther embodiments, the HAT inhibitor is selected from the groupconsisting of:(1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione(curcumin); and 2-hydroxy-6-pentadecyl-benzoic acid (anacardic acid).

One of skill in the art will recognize that agents for use within themethods of the presently disclosed subject matter include thepharmaceutically acceptable salts of the compounds described above. Theterm “pharmaceutically acceptable salts” is meant to include salts ofactive compounds, which are prepared with relatively nontoxic acids orbases, depending on the particular substituent moieties found on thecompounds described herein.

When agents for use within the methods of the presently disclosedsubject matter contain relatively acidic functionalities, base additionsalts can be obtained by contacting the neutral form of such compoundswith a sufficient amount of the desired base, either neat or in asuitable inert solvent. Examples of pharmaceutically acceptable baseaddition salts include alkali or alkaline earth metal salts including,but not limited to, sodium, lithium, potassium, calcium, magnesium andthe like, as well as nontoxic ammonium, quaternary ammonium, and aminecations, including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine and the like.

When agents for use within the methods of the presently disclosedsubject matter contain relatively basic functionalities, acid additionsalts can be obtained by contacting the neutral form of such compoundswith a sufficient amount of the desired acid, either neat or in asuitable inert solvent. Examples of pharmaceutically acceptable acidaddition salts include those derived from inorganic acids including, butnot limited to, hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids, such as acetic (acetates), propionic(propionates), isobutyric (isobutyrates), maleic (maleates), malonic,benzoic (benzoates), succinic (succinates), suberic, fumaric(fumarates), lactic (lactates), mandelic (mandelates), phthalic(phthalates), benzenesulfonic (benzosulfonates), p-tolylsulfonic, citric(citrates), tartaric (tartrates, e.g., (+)-tartrates, (−)-tartrates ormixtures thereof including racemic mixtures), methanesulfonic, and thelike. Other pharmaceutically acceptable salts, include, but are notlimited to, besylate, bicarbonate, bitartrate, bromide, calcium edetate,carnsylate, carbonate, edetate, edisylate, estolate, esylate,gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate,hydrabamine, hydroxynaphthoate, iodide, isethionate, lactobionate,malate, mesylate, mucate, napsylate, nitrate, pamoate (embonate),pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,stearate, subacetate, sulfate, tannate, and teoclate, also are included.

Also included are salts of amino acids, such as arginate and the like,and salts of organic acids, such as, glucuronic or galactunoric acids,and the like. See, for example, Berge et al, “Pharmaceutical Salts”,Journal of Pharmaceutical Science, 1977, 66, 1-19. Some compounds of thepresent disclosure can contain both basic and acidic functionalities,which allow the compounds to be converted into either base or acidaddition salts.

The neutral forms of the agents for use within the methods of thepresently disclosed subject matter may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties. For example, saltstend to be more soluble in aqueous or other protonic solvents than arethe corresponding free base forms.

In particular embodiments, the pharmaceutically acceptable salt of anagents for use within the methods of the presently disclosed subjectmatter is selected from the group consisting of HCl, a sulfonate, asulfate, phosphate, a malonate, a succinate, a fumarate, a maleate, atartrate, a 3-sulfopropanoic acid salt, and a citrate.

Certain agents for use within the methods of the presently disclosedsubject matter can exist in unsolvated forms, as well as solvated forms,including hydrated forms. In general, the solvated forms are equivalentto unsolvated forms and are encompassed within the scope of the presentdisclosure. Certain agents for use within the methods of the presentlydisclosed subject matter may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present disclosure and are intended to be within thescope of the present disclosure.

In addition to salt forms, the present disclosure provides agents foruse within the methods that can be in a prodrug form. Prodrugs of theagents for use within the methods of the presently disclosed subjectmatter are those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentdisclosure. Additionally, prodrugs can be converted to the agents foruse within the methods of the presently disclosed subject matter bychemical or biochemical methods in an ex vivo environment. For example,prodrugs can be slowly converted to the agents for use within themethods of the presently disclosed subject matter when placed in atransdermal patch reservoir with a suitable enzyme or chemical reagent.

C. Treatment Methods

As used herein, the terms “treat,” treating,” “treatment,” and the like,are meant to decrease, suppress, attenuate, diminish, arrest, theunderlying cause of a disease, disorder, or condition, or to stabilizethe development or progression of a disease, disorder, condition, and/orsymptoms associated therewith. The terms “treat,” “treating,”“treatment,” and the like, as used herein can refer to curative therapy,prophylactic therapy, and preventative therapy. The treatment,administration, or therapy can be consecutive or intermittent.Consecutive treatment, administration, or therapy refers to treatment onat least a daily basis without interruption in treatment by one or moredays. Intermittent treatment or administration, or treatment oradministration in an intermittent fashion, refers to treatment that isnot consecutive, but rather cyclic in nature. Treatment according to thepresently disclosed methods can result in complete relief or cure from adisease, disorder, or condition, or partial amelioration of one or moresymptoms of the disease, disease, or condition, and can be temporary orpermanent. The term “treatment” also is intended to encompassprophylaxis, therapy and cure.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disease, disorder, or condition in a subject, who doesnot have, but is at risk of or susceptible to developing a disease,disorder, or condition. Thus, in some embodiments, an agent can beadministered prophylactically to prevent the onset of a disease,disorder, or condition, or to prevent the recurrence of a disease,disorder, or condition.

By “agent” is meant an agent that restores balance between open andclosed chromatin states at one or more target genes, as describedelsewhere herein. In particular, the agent that restores balance betweenopen and closed chromatin states at one or more target genes is an agentthat ameliorates the effect of a defective gene encoding a component ofthe epigenetic machinery. As used herein, an agent that ameliorates theeffect of a defective gene encoding a component of the epigeneticmachinery may decrease, suppress, attenuate, diminish, arrest, orstabilize the effect of a defective gene encoding a component of theepigenetic machinery.

More generally, the term “therapeutic agent” means a substance that hasthe potential of affecting the function of an organism. Such an agentmay be, for example, a naturally occurring, semi-synthetic, or syntheticagent. For example, the therapeutic agent may be a drug that targets aspecific function of an organism. A therapeutic agent also may be anutrient. The subject treated by the presently disclosed methods intheir many embodiments is desirably a human subject, and particularly apostnatal human subject, although it is to be understood that themethods described herein are effective with respect to all vertebratespecies, which are intended to be included in the term “subject.”Accordingly, a “subject” can include a human subject for medicalpurposes, such as for the treatment of an existing disease, disorder,condition or the prophylactic treatment for preventing the onset of adisease, disorder, or condition or an animal subject for medical,veterinary purposes, or developmental purposes. Suitable animal subjectsinclude mammals including, but not limited to, primates, e.g., humans,monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like;bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and thelike; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs,and the like; equines, e.g., horses, donkeys, zebras, and the like;felines, including wild and domestic cats; canines, including dogs;lagomorphs, including rabbits, hares, and the like; and rodents,including mice, rats, guinea pigs, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a disease, disorder, orcondition. Thus, the terms “subject” and “patient” are usedinterchangeably herein. Subjects also include animal disease models(e.g., rats or mice used in experiments).

E. Dosage and Mode of Administration

Agents that restore balance between open and closed chromatin states atone or more target genes as described elsewhere herein can beadministered using a variety of methods known in the art. Theadministering can be carried out by, for example, intravenous infusion;injection by intravenous, intraperitoneal, intracerebral, intramuscular,intraocular, intraarterial or intralesional routes; or topical or ocularapplication.

More particularly, as described herein, agents within the methods of thepresently disclosed subject matter can be administered to a subject fortherapy by any suitable route of administration, including orally,nasally, transmucosally, ocularly, rectally, intravaginally,parenterally, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intra-articullar, intra-sternal, intra-synovial,intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal,or intraocular injections, intracisternally, topically, as by powders,ointments or drops (including eyedrops), including buccally andsublingually, transdermally, through an inhalation spray, or other modesof delivery known in the art. For example, for ocular administration, aneyedrop formulation can include an effective concentration of a compoundof Formula (Ia) or Formula (II) together with other components, such asbuffers, wetting agents and the like. Intravitreal injection also may beemployed to administer a presently disclosed compound to the eye.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intarterial, intrathecal,intracapsular, intraorbital, intraocular, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

For intracerebral use, agents within the methods of the presentlydisclosed subject matter can be administered continuously by infusioninto the fluid reservoirs of the CNS, although bolus injection may beacceptable. The presently disclosed compounds can be administered intothe ventricles of the brain or otherwise introduced into the CNS orspinal fluid. Administration can be performed by use of an indwellingcatheter and a continuous administration means such as a pump, or it canbe administered by implantation, e.g., intracerebral implantation of asustained-release vehicle. More specifically, the presently disclosedcompounds can be injected through chronically implanted cannulas orchronically infused with the help of osmotic minipumps. Subcutaneouspumps are available that deliver proteins through a small tubing to thecerebral ventricles. Highly sophisticated pumps can be refilled throughthe skin and their delivery rate can be set without surgicalintervention. Examples of suitable administration protocols and deliverysystems involving a subcutaneous pump device or continuousintracerebroventricular infusion through a totally implanted drugdelivery system are those used for the administration of dopamine,dopamine agonists, and cholinergic agonists to Alzheimer's diseasepatients and animal models for Parkinson's disease, as described byHarbaugh, J. Neural Transm. Suppl. 24:271, 1987; and DeYebenes et al.,Mov. Disord. 2: 143, 1987.

Agents for use within the methods of the presently disclosed subjectmatter can be manufactured in a manner known in the art, e.g. by meansof conventional mixing, dissolving, granulating, dragee-making,levitating, emulsifying, encapsulating, entrapping or lyophilizingprocesses.

More particularly, agents within the methods of the presently disclosedsubject matter for oral use can be obtained through combination ofactive compounds with a solid excipient, optionally grinding a resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients include, but are not limited to, carbohydrate or proteinfillers, such as sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethyl cellulose; and gums including arabic andtragacanth; and proteins, such as gelatin and collagen; andpolyvinylpyrrolidone (PVP:povidone). If desired, disintegrating orsolubilizing agents, such as cross-linked polyvinyl pyrrolidone, agar,alginic acid, or a salt thereof, such as sodium alginate, also can beadded to the compositions.

Dragee cores are provided with suitable coatings, such as concentratedsugar solutions, which also can contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, e.g., dosage, or different combinations ofactive compound doses.

Pharmaceutical compositions suitable for oral administration includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, e.g., a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain active ingredients admixedwith a filler or binder, such as lactose or starches, lubricants, suchas talc or magnesium stearate, and, optionally, stabilizers. In softcapsules, the active compounds can be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs), with or without stabilizers. Stabilizers can be added aswarranted.

In some embodiments, the agents for use within the methods of thepresently disclosed subject matter can be administered by rechargeableor biodegradable devices. For example, a variety of slow-releasepolymeric devices have been developed and tested in vivo for thecontrolled delivery of drugs, including proteinaciousbiopharmaceuticals. Suitable examples of sustained release preparationsinclude semipermeable polymer matrices in the form of shaped articles,e.g., films or microcapsules. Sustained release matrices includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919; EP58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers 22:547, 1983), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.15:167, 1981; Langer, Chem. Tech. 12:98, 1982), ethylene vinyl acetate(Langer et al., Id), or poly-D-(−)-3-hydroxybutyric acid (EP 133,988A).Sustained release compositions also include liposomally entrappedcompounds, which can be prepared by methods known per se (Epstein etal., Proc. Natl. Acad. Sci. U.S.A. 82:3688, 1985; Hwang et al., Proc.Natl. Acad. Sci. U.S.A. 77:4030, 1980; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324A). Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamelar type in which the lipid content isgreater than about 30 mol % cholesterol, the selected proportion beingadjusted for the optimal therapy. Such materials can comprise animplant, for example, for sustained release of the presently disclosedcompounds, which, in some embodiments, can be implanted at a particular,pre-determined target site.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of active compounds. For injection, the presentlydisclosed pharmaceutical compositions can be formulated in aqueoussolutions, for example, in some embodiments, in physiologicallycompatible buffers, such as Hank's solution, Ringer solution, orphysiologically buffered saline. Aqueous injection suspensions cancontain substances that increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds or vehicles include fatty oils, suchas sesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Optionally, the suspension also can containsuitable stabilizers or agents that increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

For nasal or transmucosal administration generally, penetrantsappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

For inhalation delivery, agents for use within the methods of thepresently disclosed subject matter also can be formulated by methodsknown to those of skill in the art, and may include, for example, butnot limited to, examples of solubilizing, diluting, or dispersingsubstances such as, saline, preservatives, such as benzyl alcohol,absorption promoters, and fluorocarbons.

Additional ingredients can be added to compositions for topicaladministration, as long as such ingredients are pharmaceuticallyacceptable and not deleterious to the epithelial cells or theirfunction. Further, such additional ingredients should not adverselyaffect the epithelial penetration efficiency of the composition, andshould not cause deterioration in the stability of the composition. Forexample, fragrances, opacifiers, antioxidants, gelling agents,stabilizers, surfactants, emollients, coloring agents, preservatives,buffering agents, and the like can be present. The pH of the presentlydisclosed topical composition can be adjusted to a physiologicallyacceptable range of from about 6.0 to about 9.0 by adding bufferingagents thereto such that the composition is physiologically compatiblewith a subject's skin.

In other embodiments, the pharmaceutical composition can be alyophilized powder, optionally including additives, such as 1 mM-50 mMhistidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5that is combined with buffer prior to use.

Regardless of the route of administration selected, agents for usewithin the methods of the presently disclosed subject matter, which maybe used in a suitable hydrated form, and/or the pharmaceuticalcompositions are formulated into pharmaceutically acceptable dosageforms such as described below or by other conventional methods known tothose of skill in the art.

The term “effective amount,” as in “a therapeutically effective amount,”of a therapeutic agent refers to the amount of the agent necessary toelicit the desired biological response. As will be appreciated by thoseof ordinary skill in this art, the effective amount of an agent may varydepending on such factors as the desired biological endpoint, the agentto be delivered, the composition of the pharmaceutical composition, thetarget tissue or cell, and the like. More particularly, the term“effective amount” refers to an amount sufficient to produce the desiredeffect, e.g., to reduce or ameliorate the severity, duration,progression, or onset of a disease, disorder, or condition (e.g.,Mendelian disorders of the epigenetic machinery), or one or moresymptoms thereof; prevent the advancement of a disease, disorder, orcondition, cause the regression of a disease, disorder, or condition;prevent the recurrence, development, onset or progression of a symptomassociated with a disease, disorder, or condition, or enhance or improvethe prophylactic or therapeutic effect(s) of another therapy.

Actual dosage levels of the active ingredients in the presentlydisclosed pharmaceutical compositions can be varied so as to obtain anamount of the active ingredient that is effective to achieve the desiredtherapeutic response for a particular subject, composition, route ofadministration, and disease, disorder, or condition without being toxicto the subject. The selected dosage level will depend on a variety offactors including the activity of the particular compound employed, orsalt thereof, the route of administration, the time of administration,the rate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compound employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the agents for use within the methods of the presentlydisclosed subject matter at levels lower than that required to achievethe desired therapeutic effect and gradually increase the dosage untilthe desired effect is achieved. Accordingly, the dosage range foradministration will be adjusted by the physician as necessary. It willbe appreciated that an amount of a compound required for achieving thedesired biological may be different from the amount of compoundeffective for another purpose.

In general, a suitable daily dose of an agent for use within the methodsof the presently disclosed subject matter will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. Generally, doses of the agents for use within themethods of the presently disclosed subject matter will range from about0.0001 to about 1000 mg per kilogram of body weight of the subject perday. In certain embodiments, the dosage is between about 1 μg/kg andabout 500 mg/kg, more preferably between about 0.01 mg/kg and about 50mg/kg. For example, in certain embodiments, a dose can be about 1, 5,10, 15, 20, or 40 mg/kg/day. If desired, the effective daily dose of anagent for use within the methods of the presently disclosed subjectmatter can be administered as two, three, four, five, six or moresub-doses administered separately at appropriate intervals throughoutthe day, optionally, in unit dosage forms.

II. General Definitions

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, parameters,quantities, characteristics, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about” even though the term “about” may notexpressly appear with the value, amount or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are not and need not beexact, but may be approximate and/or larger or smaller as desired,reflecting tolerances, conversion factors, rounding off, measurementerror and the like, and other factors known to those of skill in the artdepending on the desired properties sought to be obtained by thepresently disclosed subject matter. For example, the term “about,” whenreferring to a value can be meant to encompass variations of, in someembodiments, ±100% in some embodiments ±50%, in some embodiments ±20%,in some embodiments ±10%, in some embodiments ±5%, in some embodiments±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1 Materials and Methods

Study Design:

The purpose of this study was to explore the pathophysiological sequencein KS, a Mendelian disorder of the epigenetic machinery, and to seekrobust disease associated phenotypes, which could be used to monitortherapeutic response. It was hypothesized that since both causes of KSinvolve the transition from closed to open chromatin, this disordermight be caused by a general imbalance between open and closed chromatinstates (favoring closed chromatin) and this ongoing deficiency might beameliorated with agents that favor chromatin opening such as HDACi. Atleast 3-4 biological replicates were used for each biochemical analysis,while a sample size of at least 8-10 per group was used for behavioraltesting. Data collection occurred for a pre-determined and of consistentduration, as dictated by literature-based or core facility-basedstandards and no exclusion criteria were applied. All analyses wereperformed by examiners blinded to genotype and/or treatment arm. Fordrug treatments, animals were randomly assigned to treatment arms withapproximately equivalent numbers in each group. Box and whisker plotsidentify RStudio-defined outliers (shown as circles), but all datapoints were used in statistical analyses.

Design of the Indicator Constructs.

A genetically encoded histone reporter allele system has been developedwhich can be used to monitor activity of any histone maintenancemachinery component in live cells. Previously, FRET based epigeneticactivity systems have been created (Lin and Ting (2004) Angew Chem IntEd Engl. 24; 43 (22):2940-3; Lin et al. (2004) J. Am. Chem. Soc. 19;126(19):5982-3; U.S. Pat. No. 7,056,683). However, FRET-based assaysneed a complex technological setup which is not widely available andFRET-based assays have been much more difficult to introduce intotransgenic mouse models. Here, the presently disclosed non-FRET-basedhistone indicator system is demonstrated through examples of two wellunderstood histone modifications, histone acetylation and histone H3K4trimethylation. The particular construct design is based on a circularlypermutated green fluorescent protein (GFP) that lacks fluorescenceunless the two parts of GFP are brought into close proximity by externalforces (Baird et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 28;96(20):11241-6). The constructs are targeted to the nucleus through anuclear localization signal (NLS). Mutants were also created thatknocked out the activity of the indicator allele.

The acetyl reporter protein quantifies the activity of the acetylationmachinery (acetylation of H4 specifically at the 5th, 8th, 12th and 16thLysine's) and comprises an H4 tail on one end (the target foracetylation) and a TBP associated factor II (TAFII) bromodomain on theother end of the reporter protein (FIG. 5A). The TAFII bromodomain onlyrecognizes and binds to the acetylated H4 tail, resulting inreconstitution of GFP structure and function (i.e. fluorescence).Therefore, the reporter protein has no fluorescence unless it isacetylated by the acetylation system of the nucleus of the cell.

An H3K4 trimethylation reporter was also created (FIG. 5B). Thisreporter is based on the H3 tail on one end and the TBP associatedfactor III (TAFIII) homeodomain on the other end; the TAFIII homeodomainonly identifies and binds to trimethylated K4 on H3. When the H3K4 sitegets trimethylated, the TAFIII homeodomain can bind to the modified H3tail and bring the two parts of the separated GFP in close proximity.The activity of the epigenetic modification system can be quantifiedthrough fluorescence.

Epigenetic Reporter Alleles:

Epigenetic reporter alleles were synthesized (OriGene, Rockville, Md.)using published sequences for component elements (Baird et al. (1999)Proc. Natl. Acad. Sci. 96:11241-6; Souslova et al. (2007) BMCBiotechnol. 7:37). Single nucleotide mutations were created using theQuickChange Lightening kit (Agilent Technologies Inc, Santa Clara,Calif.). For H4ac indicator, K5R, K8R, K12R, K16R and K20R (MUTindicator) were introduced. For H3K4me3 indicator, K4Q and D890A/W891Aand M882A (three separate constructs) were introduced. For transienttransfections, mouse embryonic fibroblasts (see below) were transfectedwith Fugene HD (Promega, Madison, Wis.), 48 hours prior to FACS.Transfection efficiency of reporter alleles was comparable intransiently-transfected murine embryonic fibroblasts (MEFs) derived frommice of both genotypes (Kmt2d^(+/βGeo) and Kmt2d^(+/+)), as measured byreal-time PCR of genomic DNA. For drug stimulation, drug was added tothe media 24 hours prior to FACS. For stable transfections in T293(American Type Culture Collection) cells, 10 μg/ml of Blastocidin (LifeTechnologies, Carlsbad, Calif.) was added to the media for severalweeks. For stable transfection in MEFs, the reporter was transferred toa ViraPower Lentiviral Expression System (Life Technologies, Carlsbad,Calif.). After selection with Blastocidin, the drug of interest wasadded 24 hours prior to FACS. SAHA, AR-42 and MS275 were purchased fromSelleck (Selleck Chemicals, Houston, Tex.). FACS was performed usingeither a FACSCalibur (BD Biosciences, San Jose, Calif.) or FACSverse (BDBiosciences, San Jose, Calif.) system with comparable results. FACS datawere analyzed using FlowJo (Tree Star Inc, Ashland, Oreg.). A plasmidexpressing HDAC3 was acquired from Addgene (Cambridge, Mass., plasmid13819) and transfected into a stable cell line carrying the H4 acetylreporter allele.

Animals:

Kmt2d^(+/βGeo) mice, also named Mll2Gt^((RRt024) Byg), were acquiredfrom Bay Genomics (University of California). All experimental mice wereon a mixed C57BL/6J and 129/SvEv background. Expected Mendelian ratioswere observed when heterozygous animals were bred to wild-type. Inheterozygous crosses, however, there was uniform embryonic lethality ofhomozygotes by ED 12, the earliest developmental stage assayed. Fortreatment with AR-42, mice were orally gavaged daily with drug (SelleckChemicals, Houston, Tex.) solubilized in vehicle (0.5% methylcellulose,0.1% Tween-80, water) or with vehicle alone. Drug delivery informationwas kindly provided by Drs. Chen and Kulp from Ohio State University(Huang et al. (2011) Mol. Pharmacol. 79:197-20619). Drug wasadministered for 14 days and mice were sacrificed on day 15. Morriswater maze testing was initiated at day 7 and a dose of 10 mg/kg/day wasused for these studies. For quantification of DCX positive cells, dosesof 0, 5, 10 and 25 mg/kg/day were used. Genotyping was performed usingprimers B-GeoF-(CAAATGGCGATTACCGTTGA; SEQ ID NO: 1) andB-GeoR-(TGCCCAGTCATAGCCGAATA; SEQ ID NO: 2) that are specific for thetargeted allele and TcrdF-(CAAATGTTGCTTGTCTGGTG; SEQ ID NO: 3) andTcrdR-(GTCAGTCGAGTGCACAGTTT; SEQ ID NO: 4) that control for sufficientDNA concentration. Real-time PCR using the same primers allowsdiscrimination between the heterozygous and homozygous state for thetargeted allele. Maxillary protrusion was evaluated by ten investigatorsblinded to genotype and they were asked to rate maxillary protrusion onradiographs as either large (2) or small (1). When results wereunblinded and average scores for each animal determined, theKmt2d^(+/βGeo) animals had a significantly lower score than Kmt2d^(+/+)littermates. FIG. 30 shows the methyl-indicator in murine embryonicfibroblasts (MEFs) from the Kabuki syndrome mouse model (M112+/−) versusfrom wild type mice. All experiments were performed using mouseprotocols approved by the Animal Care and Use Committee of Johns HopkinsUniversity School of Medicine. The mouse protocols used for this studyare in accordance with the guidelines used by the NIH for mouse care andhandling.

Perfusion and Cryosectioning:

Mice were sacrificed with a xylazine ketamine combination,transcardially flushed with PBS/Heparin and then perfused with 4%PFA/PBS. Brains were dissected and cryopreserved in 30% sucrose 0.1Mphosphate solution overnight at 4° C. Brains were frozen and sectionedusing a Microm HM 550 cryostat (Thermo Scientific, Waltham, Mass.).Sectioning was performed at 30 μm intervals and every section of thebrain was collected and stored in glycerine ethelyne glycol phosphatestorage solution.

EdU Administration and Staining:

For EdU experiments, mice were injected IP over ten days, withinjections on the first three and last three days, with 50 mg/kg EdU(Life Technologies, Carlsbad, Calif.). Mice were sacrificed 30 daysafter the initial start of injection, and EdU staining was done with theClick-iT EdU Alexa Fluor 488 Imaging Kit (Life Technologies, Carlsbad,Calif.) as well as DAPI mounting with Vectamount (Vector Laboratories,Burlingame, Calif.). EdU quantification was performed by an individualblinded to genotype. Positive cells were counted in every sixth slice inthe GCL and the average number per slice was calculated for each brain.

Real Time PCR:

Real-time PCR was performed using Kmt2d-specific probes for exons 20 and52 (Mm_02600438 and Mm_01717664, respectively) from TaqMan® GeneExpression Assays (Life Technologies, Carlsbad, Calif.). For acomparison of transfection efficiencies for indicator constructs intransient transfection studies, real-time PCR was performed using SYBRGreen Real-Time PCR Master Mix (Life Technologies, Carlsbad, Calif.) andprimers IND-F-(CTGCGCGCAAGTCAACGGGTG; SEQ ID NO: 5) andIND-R-(ATGCCGTTCTTCTGCTTGTCG; SEQ ID NO: 6) that are specific for theH3K4 methylation indicator. For expression analysis for KMT2D targetgene KLF10, real-time PCR was performed using Klfl0-specific expressionassay (Mm00449812_m1) from TaqMan® Gene Expression Assays (LifeTechnologies, Carlsbad, Calif.).

Immunoblotting:

Total protein lysates from Kmt2d^(+/βGeo) and Kmt2d^(+/+) littermateswere isolated and immunoprecipitated with an antibody against the aminoterminus of KMT2D (sc-292359, Santa Cruz Biotechnology, Dallas, Tex.).Isolated protein was applied to a membrane and immunoblotted with anantibody against beta-galactosidase (ab9361, Abcam, Cambridge, ENG) aspreviously described (Loeys et al. (2010) Sci. Transl. Med. 23: 23ra20).

Immunofluorescence:

Every 6^(th) brain section was selected and then blocked with 5% BovineSerum Albumin (BSA) at room temperature followed by incubation withprimary antibodies overnight at 4° C. Secondary antibodies were thenapplied for 1 hour at room temperature, after which sections weremounted onto microscope slides with Vectamount with DAPI (VectorLaboratories, Burlingame, Calif.). Primary antibodies includedDoublecortin (DCX) (SC-8066, Santa Cruz Biotechnology, Dallas, Tex.,1:200 goat), trimethylated H3K4 (9727L, Cell Signaling Technology,Beverly, Mass., 1:500 rabbit), and Kmt2d H-300 (SC-292359, Santa CruzBiotechnology, Dallas, Tex., 1:500 rabbit). Non-specific binding was notobserved when sections were sequentially exposed to serum (or IgG whenappropriate) from the same species as the primary antibody for eachexperiment (i.e. rabbit for KMT2D and H3K4me3 and goat fordoublecortin), followed by the secondary antibody used for KMT2D andH3K4me3 (anti-rabbit) or doublecortin (anti-goat, FIG. 29).

Confocal Microscopy:

Z-stack images of slides were taken at either 10× using Zeiss Axiovert200 with 510-Meta confocal module (Carl Zeiss, Jena, GER) or 25× usingZeiss AxioExaminer with 710NLO-Meta multiphoton (Carl Zeiss, Jena, GER).From 10× pictures, the GCL was highlighted and fluorescent intensitiesfor both DAPI and H3K4me3 were measured at the midpoint of the entirez-stack (Zen software, Carl Zeiss, Jena, GER) with the value forKmt2d^(+/+) animals set equal to one. A Students t-test withsignificance value set at P<0.05 was used to compare H3K4 trimethylationintensity referenced to DAPI intensity.

GCL and Doublecortin Area:

The area of both the GCL and DCX+ cells was measured using the NSelements 2.0 software (Nikon, Tokyo, JPN). Normalized DCX area wascalculated by measuring the DCX+ area of the GCL and setting thebaseline (Kmt2d^(+/+)) fraction to 1. A Student t-test with significancevalue set at P<0.05 was used for comparison of DCX+ area referenced toGCL area between genotypes and treatment arms.

Behavioral Testing:

Mice ranged from two to three months of age in all tests, and allexperiments were performed in the late morning or early afternoon.

Novel Object Recognition:

On the first day of the novel object recognition test, mice wereindividually placed into a square plastic arena (25 cm×25 cm×25 cm) thatcontained two identical plastic objects along the midline of the arena.Each mouse was allowed to explore the objects for 10 minutes and thenplaced back in its home cage. The following day, each mouse was placedin the same arena with the same two identical objects and the timeinteracting with each object was recorded over 10 minutes. On the thirdday, one object was removed and was replaced by a novel object. Micewere placed in the arena for five minutes and timed for interaction withthe familiar object compared to the novel object. Interactions withobjects were recorded and measured in a way that was blinded to bothgenotype and drug treatment. Differences in interaction time between thenovel object and the familiar object for Kmt2d^(+/+) and Kmt2d^(+/βGeo)mice were calculated by computing time spent with the novel objectdivided by the total time spent with both objects. These values wereanalyzed for significance with a Student's t-test with significancevalue set at P<0.05.

Morris Water Maze:

Mice were placed in a 1.1 meter diameter tank filled with roomtemperature water dyed with non-toxic white paint. For analysispurposes, the tank was divided into four quadrants, with one quadrantcontaining a small platform submerged 1.5 cm beneath the water. On eachday of training, mice were placed in the tank in a random quadrantfacing away from the center and were allowed to swim until they foundthe platform and were left there for 30 seconds. If they did not reachthe platform after 60 s they were placed on it for 30 seconds. Eachmouse was given 4 trials per day (for 5 days) with no inter-trialinterval and subsequently returned to its home cage. Latency to reachthe platform was measured during each trial. The day after the final dayof training, the platform was removed for a probe trial where mice wereplaced in the tank for 90 s. Average number of crossings of theplatform's previous location was recorded. Visible/flagged platformtraining was also performed for 3 days either before the hidden platformor after the probe trial, where a visible flag was placed on thesubmerged platform, and the time for each mouse to reach the platformwas measured for each 60 second trial, four of which were run in thesame way as the hidden platform training. For all training and probetesting, data was recorded both manually and electronically withANY-maze software (San Diego Instruments, San Diego, Calif.) whenapplicable. All data were collected and analyzed by an individualblinded to genotype and treatment group. Differences in the number ofplatform crossings during the probe trial were compared between groupswith a Student's t-test with significance value set at P<0.05.

Fear Conditioning Testing:

On day 1, both Kmt2d^(+/ρGeo) mice and Kmt2d^(+/+) littermates wereplaced in chamber and allowed to explore the chamber freely. After 120seconds (2 minutes), a 2000 Hz sound was played for 30 seconds. For thelast 2 seconds of sound (seconds 148-150), the sound co-occurred with a0.35 mAmp electrical shock (2 seconds) administered through the floorgrid. Mice were observed for a total of 300 seconds. Freezing behaviorwas measured using the FreezeScan software (CleverSys Inc, Reston, Va.).On days 2 and 14 (FIG. 9), contextual freezing was assessed over 300seconds (no cue). On days 3 and 15, cued freezing was assessed over 300seconds.

Open Field Testing:

Mice were placed in the open field chamber and activity was monitoredusing the Photobeam activity system (San Diego Instruments, San Diego,Calif.). Activity levels (ten 180 second intervals) were pooled to yielda general activity level (Adamczyk et al. (2012) Behav Brain Res. 229:265-72).

Grip Strength Testing:

Grip strength testing was performed as previously described (Adamczyk etal. (2012) Behav Brain Res. 229: 265-72). Three trials were performedand averaged for each mouse.

Retrospective Analysis of Neuropsychological Testing on Patients withKabuki Syndrome:

A retrospective chart review was performed using data from patients thathad clinically indicated neuropsychological testing at the KennedyKrieger Institute in years 2004-2014. Test results were analyzed fromthe three individuals with most extensive testing available and a knowndisease associated mutation in KMT2D. All patient data was collectedafter consenting patients and stored in secure electronic database atKKI. For this particular analysis per Kennedy Krieger and Johns Hopkinsorganizational policy, additional IRB review was not required (three orfewer patients). The individual tasks were divided into 16 categories,and literature was used to identify tasks known to be associated withdentate gyrus (Kesner (2013) Behav. Brain Res. 254:1-7; Morris et al.(2012) Neurobiol. Learn. Mem. 97:326-31; Epp et al., (2011) Neurobiol.Learn. Mem. 95:316-25) or hippocampus (non-dentate gyrus).

ChIP-seq:

Spleens were dissected from eight mice, four from each kmt2d genotype(+/βGeo or +/+) where half of each genotype was treated with AR-42 andhalf with vehicle only. Spleens were minced and passed through a 40 mcell strainer to obtain single cell suspensions. 10 million cells wereused for each ChIP-seq experiment following the native chromatinimmunoprecipitation protocol, as previously described (Gilfillan et al.(2012) BMC Genomics 13:645), using a ChIP-grade antibody against H3K4me3(9727, Cell Signaling Technology, Beverly, Mass.).

ChIP-Seq Data Analysis:

Sequencing was performed using a MiSeq system (Illumina, San Diego,Calif.). 4.8-9.6 million paired-end 26 bp reads were obtained per sample(Table 2; nReads=number of reads, alignRate=fraction aligned to genome;FRIP=fraction of reads in peaks). Reads were aligned to the m. musculusgenome, version mm 10, using Bowtie 2 (Langmead and Salzberg (2012) Nat.Methods. 9: 357-9). Each sample was examined with regard to alignmentrate as well as FRIP (fraction of reads in peaks), a measure of the ChIPefficiency (Table 2). FRIP was computed based on peaks called only onspecific samples using MACS version 1.4.2 (Zhang et al. (2008) GenomeBiol. 9: R137). For analysis, reads were merged into one meta-sample andpeak calling was performed using MACS version 1.4.2 (Zhang et al. (2008)Genome Biol. 9: R137). This allowed definition of a superset of 33,517peaks in one or more samples. The number of reads overlapping a peak wascomputed using bedtools version 2.17.0 (Quinlan and Hall (2010)Bioinformatics 26:841-2) in the following way: each paired-end read wasconverted to a single interval containing both mate coordinates(effectively filling in the insert) and these intervals were examinedfor overlaps with the superset of peaks. This created a peak by samplematrix of read counts. Differential binding was assessed using the GLMfunctionality (McCarthy et al. (2012) Nucleic Acids Res. 40:4288-97) inedgeR version 3.5.27 (Robinson et al. (2010) Bioinformatics. 26:139-40).A single model was fit, using all 8 samples, with Tagwise varianceestimation. Different contrasts were examined corresponding to thedifferent hypotheses considered in the main text, and peaks wereconsidered differentially bound if they had a Benjamini-Horchbergcorrected p-values less than 5%. Fold change and overall abundance wascalculated as per edgeR.

For FIG. 6D, FIG. 25E, FIG. 27A, FIG. 27B, FIG. 27C, FIG. 27D, and FIG.27E, each point corresponds to a genomic location with an H3K4me3 peakin at least one of the samples. A positive value indicates that the peakis higher in the Kmt2d^(+/βGeo) compared to the Kmt2d^(+/+). Peaks whichare significantly differentially bound are shown in red, and other peaksare shown in gray. In FIG. 27A, FIG. 27B, FIG. 27C, FIG. 27D, and FIG.27E, the expected medium is demonstrated with a broken line, butunbroken line shows the median in the observed comparison. FIG. 28A,FIG. 28B, FIG. 28C, FIG. 28D and FIG. 28E illustrate whether the balanceis shifting up (blue) or down in a particular comparison. To examinewhether there was a directional balance between differentially boundgenes, the following test was developed. For Kmt2d^(+/+) (vehicle)compared to Kmt2d^(+/βGeo) (vehicle), 454 peaks are observed to bestronger bound in the mutant, 1,499 to be stronger bound in thewild-type and 27,052 peaks to not be differentially bound. The modelassumes that these three numbers follow a multinomial distribution witha probability vector (p1, p2, p3). To test for directional balance, astandard likelihood-ratio test for the hypothesis p1=p2 is constructed.Per standard results, two times the negative log-likelihood ratio teststatistic is asymptotically chi-square distributed with 1 degree offreedom.

TABLE 2 A summary of genotypes, drugs and quality measures of ChIP-seqexperiments. Genotype Drug Run nReads alignRate FRIP Kmt2d^(+/βGeo)AR-42 run4 5690117 0.92 0.508 Kmt2d^(+/βGeo) Vehicle run4 7520215 0.930.556 Kmt2d^(+/+) AR-42 run4 9614420 0.92 0.48 Kmt2d^(+/+) Vehicle run46828962 0.92 0.571 Kmt2d^(+/βGeo) AR-42 run5 7604137 0.93 0.53Kmt2d^(+/βGeo) Vehicle run5 6016687 0.92 0.55 Kmt2d^(+/+) AR-42 run54846490 0.93 0.545 Kmt2d^(+/+) Vehicle run5 8682723 0.93 0.573

Statistics and Plots:

For all box plots generated through RStudio (RStudio Inc, Boston,Mass.), the margins of the box show the upper and lower quartiles, thecentral line shows the median, and the whiskers show the range. Circlesdenote outliers as defined by the RStudio algorithm. For all column,line, and scatter-plot graphs (generated through Microsoft Excel), theerror bars represent standard error of the mean, with the data pointrepresenting the mean of each applicable group. Unless otherwise stated,significance between two groups was calculated with a Student's t-testwith a significance value of P<0.05. Two-way repeated measures ANOVAswere calculated with SPSS (IBM, Armonk, N.Y.). For every calculated Pvalue the stated n represents the number of animals for each groupcontributing to that comparison. For P value nomenclature, *=P<0.05,**=P<0.01, t=P<0.005, ^(††)=P<0.001.

Results

Kmt2d^(+/βGeo) Mice:

KMT2D is a member of the mixed lineage leukemia (MLL) family ofDrosophila Trithorax orthologs that is encoded on human chromosome 12and mouse chromosome 15. An alternative name for KMT2D is mixed lineageleukemia 2 (MLL2). All members of this family contain a SET domain,which confers the H3K4 methyltransferase activity, as well as otherdomains (Hunter et al. (2012) Nucleic Acids Res. 40: 306-12) thatdelineate individual functions (FIG. 6A). A mouse model harboring aloss-of-function allele for Kmt2b, encoded on human chromosome 19 andmouse chromosome 7, has been characterized previously (Kerimoglu et al.(2013) J. Neurosci. 33, 3452-64), demonstrating hippocampal memorydefects. This gene has been alternatively designated M114 or M112,leading to confusion in the literature regarding nomenclature for thisparticular gene family, as discussed in a recent publication byBögerhausen et al. ((2013) Clin. Genet. 83: 212-4). To specificallyassess the underlying pathogenesis of KS, a novel mouse model has beencharacterized with insertion of an expression cassette encoding aβ-galactosidase neomycin resistance fusion protein (β-Geo) into intron50 of Kmt2d (Mll2) on mouse chromosome 15. Inclusion of a spliceacceptor sequence and a 3′-end cleavage and polyadenylation signal atthe 5′ and 3′ ends of the β-Geo cassette, respectively, is predicted togenerate a truncated KMT2D protein with peptide sequence correspondingto the first 50 exons of Kmt2d fused to β-Geo, but lacking the SETdomain and therefore methyltransferase activity (FIG. 6B and FIG. 7A).As predicted from this targeting event, quantitative real-timepolymerase chain reaction analysis of Kmt2d messenger RNA inKmt2d^(+/βGeo) mice demonstrates normal abundance of sequencecorresponding to exon 20 but a 50% reduction for exon 52, when comparedto Kmt2d^(+/+) littermates (FIG. 6C). Expression of aKMT2D-β-galactosidase fusion protein in Kmt2d^(+/βGeo) animalsdemonstrates transcription and translation of the targeted allele (FIG.7B). Furthermore, chromatin immunoprecipitation followed by nextgeneration sequencing (ChIP-seq) on splenic cells from Kmt2d^(+/βGeo)mice and Kmt2d^(+/+) littermates using an antibody against H3K4me3reveals an overall genome-wide decrease in H3K4me3 in Kmt2d^(+/βGeo)mice (FIG. 6D), supporting the predicted functional consequences of themutant allele. Finally, Kmt2d^(+/βGeo) mice demonstrate facial featuresthat are consistent with KS including flattened snout (FIG. 8A) anddownward rotation of the ear canal (FIG. 8B). Blinded analysis of X-raysof Kmt2d^(+/βGeo) mice revealed a significantly shorter maxilla(P<0.005) when compared to Kmt2d^(+/+) littermates (FIG. 8B and FIG.8C), as judged by the extent of protrusion beyond the mandible (FIG.8C). Kmt2d^(+/βGeo) mice demonstrate hippocampal memory defects:Disruption of several histone modifying enzyme genes has been shown tolead to hippocampal memory defects in mice, illustrating a critical rolefor epigenetic homeostasis in memory acquisition (Guan et al. (2002)Cell. 111: 483-93; Gupta et al. (2010) J. Neurosci. 30: 3589-99;Cohen-Armon et al. (2004) Science 304:1820-2). Kmt2d^(+/βGeo) mice showsignificant deficits in novel object recognition, (FIG. 6E), Morriswater maze tests (FIG. 6F) and contextual fear conditioning (FIG. 9)when compared to Kmt2d^(+/+) littermates, all consistent withhippocampal memory dysfunction. When performed before the hiddenplatform stage of training, the flag-training phase of the Morris watermaze did not reveal significant differences between Kmt2d^(+/βGeo) andKmt2d^(+/+) littermates (FIG. 10). Importantly, Kmt2d^(+/βGeo) mice didnot show decreased activity (FIG. 11A), reduced grip strength (FIG. 11B)or slower swim speeds (FIG. 11C), any of which would be indicative of amore generalized limitation of performance potential in these assays.There were no significant differences in the time that it tookKmt2d^(+/βGeo) mice to identify the platform (escape latency) comparedto Kmt2d^(+/+) mice during the training phase (FIG. 12A, FIG. 12B, FIG.12C, and FIG. 12D).

Decreased Dentate Gyrus Volume and Defective Neurogenesis inKmt2d^(+/βGeo) Mice:

Immunofluorescence analyses revealed particularly high levels ofexpression of KMT2D protein in the dentate gyrus GCL of the hippocampusin Kmt2d^(+/+) mice (FIG. 13A) and a striking deficiency of H3K4me3 inthe dentate gyrus GCL of Kmt2d^(+/βGeo) mice compared to wild-type (WT)littermates (FIG. 13B and FIG. 13C). A similar deficiency of H3K4me3 wasalso seen in the pyramidal layer of the hippocampus (FIG. 14). Thelevels of H3K4me3 showed substantial cell-to-cell variability inKmt2d^(+/βGeo) animals (FIG. 13B), suggesting that variation in cellstate or identity within the GCL or dentate gyrus might influencevulnerability to the consequences of heterozygous Kmt2d disruption.Kmt2d^(+/βGeo) mice showed a significant decrease in body but not brainweight (FIG. 15A and FIG. 15B), and had reduced dentate gyrus GCL volumewhen standardized to brain weight (FIG. 13D and FIG. 13E). Thiscorrelated with reduced neurogenesis in the GCL of Kmt2d^(+/βGeo) mice,as evidenced by significantly reduced expression of both doublecortin(DCX; Rao and Shetty (2004) Eur. J. of Neurosci. 19: 234-246) (FIG. 13Fand FIG. 13G) and 5-ethynyl-2′-deoxyuridine (EdU) staining, a marker ofboth neurogenesis in the GCL and a marker of neuronal survival whenmonitored 30 days after labeling (FIG. 16). Confocal microscopy revealedan apparent decrease in dendritic branching complexity of DCX positivecells (DCX+) in the GCL of Kmt2d^(+/βGeo) mice (FIG. 17). However, giventhe decreased amounts of DCX+ cells in these mice, further work isneeded to determine if this is a true or primary manifestation of Kmt2ddeficiency. To explore whether there are hippocampal memory defects inpatients with KS, comprehensive neuropsychological testing performed onthree patients with known disease causing mutations in KMT2D wasanalyzed (Table 3; N/A=not adequately tested with utilized testingregimen; ⬇=deficient area (defined as >1 standard deviation below themean and lower than full scale IQ or, if unavailable, highest individualtest score; metrics linked to the dentate gyrus are indicated in yellow(Kesner (2013) Behav. Brain Res. 254:1-7; Morris et al. (2012)Neurobiol. Learn. Mem. 97:326-31; Epp et al., (2011) Neurobiol. Learn.Mem. 95:316-25); metrics more broadly linked to the hippocampus areindicated with an asterisk (Brickman et al. (2011) Hippocampus.21:923-8)). Although, not all deficiencies observed can be explained byhippocampal dysfunction, patients consistently had abnormalities oftasks known to be associated with dentate gyrus function (Kesner (2013)Behav. Brain Res. 254:1-7; Morris et al. (2012) Neurobiol. Learn. Mem.97:326-31; Epp et al., (2011) Neurobiol. Learn. Mem. 95:316-25). Otherfunctions linked to other regions of the hippocampus (Brickman et al.(2011) Hippocampus. 21:923-8) were also abnormal in some patients aswere some tasks not linked to hippocampus indicating that other cellpopulations in the central nervous system may also play a role. Thesedata support the hypothesis that observations in Kmt2d^(+/βGeo) miceare, at least in part, reminiscent of those seen in KS.

TABLE 3 A retrospective analysis of neuropsychological testing on threepatients with mutations in KMT2D reveals consistent abnormalities offunctions that have been associated with the dentate gyrus.Neuropsychological testing of patients with known disease causingmutations in KMT2D. Patient 1 Patient 2 Patient 3 Neuropsychologic 28yrs 15 yrs 14 yrs process/function Female Female Male Affected GeneKMT2D KMT2D KMT2D Full Scale IQ 87 84 66 Perceptual or Non- ↓ ↓ ↓ verbalReasoning* Verbal Reasoning/ Normal Normal ↓ Comprehension VerbalFluency* ↓ Normal N/A Naming* Normal Normal Normal Vocabulary/ NormalNormal N/A Reading Processing Speed ↓ ↓ ↓ Basic Math Normal ↓ N/ACalculation Visual Selective ↓ ↓ N/A Attention* Visual Working ↓ ↓ ↓Memory* Verbal Working Normal Normal ↓ Memory* Visual Delayed ↓ ↓ ↓Memory* Verbal Delayed ↓ ↓ Normal Memory* Switching/ ↓ ↓ N/A InhibitionVerbal Organization Normal Normal N/A Visual ↓ ↓ ↓ Organization* FineMotor ↓ ↓ ↓

Application of Reporter Alleles for Epigenetic Modifications inEmbryonic Fibroblasts from Kmt2d^(+/βGeo) Mice:

Epigenetic reporter systems were created that monitor either H4acetylation or H3K4 trimethylation machinery activity in an effort todetermine whether there is an ongoing activity deficiency in cells fromKmt2d^(+/βGeo) mice (FIG. 18A). Both reporter alleles encode halves ofgreen fluorescent protein separated by a flexible linker region (Bairdet al. (1999) Proc. Natl. Acad. Sci. 96: 11241-6) with a histone tailand a histone reader at the N- and C-termini, respectively. When thehistone tail corresponding to either H4 or H3 is modified by acetylationor methylation, respectively, GFP structure and function arereconstituted, as detected by a fluorescent readout (FIG. 18B). Theacetyl reporter protein quantifies the activity of the acetylationmachinery (acetylation of H4 specifically at sites K5, K8, K12, and K16)and comprises an H4 tail (residues 1-30) on one end and a TATA bindingprotein (TBP)-associated factor II (TAFII) bromodomain on the other end(FIG. 18A). The TAFII bromodomain only recognizes and binds to theacetylated H4 tail. This acetylation-dependent reporter proteindemonstrates a linear fluorescence response when quantified byfluorescence-activated cell sorting (FACS) in the presence of increasingamounts of suberoylanilide hydroxamic acid (SAHA), an HDACi, in culturesystems (FIG. 18C, FIG. 18D, FIG. 19A, and FIG. 19B). For example, only5% of cells were easily discriminated from auto fluorescence with 1 μMof SAHA, but increased to 20% with 2.5 μM of SAHA, 40% at 7.5 μM of SAHAand 45% at 1 μM of SAHA (FIG. 18D). Saturation of this responsecorrelates well with immunoblot data using antibodies to the modified H4tail (Munshi et al. (2006) Mol. Cancer Ther. 5:1967-74). This responseis attenuated by co-transfection with a construct encoding a histonedeacetylase (FIG. 20) and absent upon mutagenesis of all potentialacetylation sites (FIG. 18E and FIG. 18I), attesting to its specificity.The H3K4 trimethylation reporter allele encodes the H3 tail (residues1-40) on one end and the TBP-associated factor III (TAF3) planthomeodomain (PHD) on the other end, which binds to trimethylated K4 onH3 (FIG. 18A). The H3K4 trimethylation reporter also demonstrates a doseresponse with increasing levels of the HDACi AR-42 (FIG. 18F), inkeeping with prior work suggesting that AR-42 can also influence themethylation status of H3K4 through inhibition of demethylases (Huang etal. (2011) Mol. Pharmacol. 79:197-206). Activity is greatly attenuatedupon mutagenesis of critical residues (M882A, W891A; Vermeulen et al.(2007) Cell 131, 58-69; van Ingen et al (2008) Structure 16, 1245-56) inthe TAF3 reader domain (FIG. 18G) or with mutation of K4 (H3K4Q) in theH3 tail (FIG. 18G). Both reporter alleles show decreased activity whenstably introduced into embryonic fibroblasts derived from Kmt2d^(+/βGeo)mice, when compared to Kmt2d^(+/+) littermates (FIG. 21). H3K4trimethylation activity is enhanced upon treatment of Kmt2d^(+/βGeo)cells with HDAC inhibitors AR-42 or MS275 (FIG. 18H, FIG. 22A, and FIG.23). An analysis of transfection efficacy in cells with both genotypesindicated comparable transfection efficacy (FIG. 22B).

Impaired Neurogenesis and H3K4 Trimethylation Deficiency inKmt2d^(+/βGeo) Mice is Rescued Upon Treatment with the HDACi AR-42:

Because of the ability of HDACi to increase H3K4 trimethylation in vitroin Kmt2d^(+/βGeo) cells, it was next asked whether the H3K4trimethylation deficiency seen in the dentate gyrus GCL ofKmt2d^(+/βGeo) mice could be attenuated or reversed upon in vivopostnatal treatment with an HDACi. Previously, the HDACi's AR-42 andMS275 have both been shown to increase H3K4 trimethylation and histoneacetylation (Huang et al. (2011) Mol. Pharmacol. 79:197-206). AR-42appeared to have the strongest effect on H3K4me3 and was thereforechosen for in vivo studies (Huang et al. (2011) Mol. Pharmacol.79:197-206). An AR-42 dose of 25 mg/kg/day was started, previously usedin mouse models of prostate cancer (Huang et al. (2011) Mol. Pharmacol.79:197-206), commencing at 20 weeks of age and continuing for two weeks.This dose increased H3K4 trimethylation in the GCL in Kmt2d^(+/βGeo)mice, compared to untreated mutant littermates (FIG. 24A and FIG. 24B),to a level that was indistinguishable from treated Kmt2d^(+/+) animals.Unexpectedly, however, this dose of AR-42 was associated with decreasedDCX expression in the GCL in both young (1-2 month-old) and old (5-6month-old) Kmt2d^(+/+) and Kmt2d^(+/βGeo) mice (FIG. 24C and FIG. 24D).Given the known cytotoxic potential of AR-42 (Huang et al. (2011) Mol.Pharmacol. 79:197-206; Zhang et al. (2011) Int. J. Cancer. 129:204-13),5 and 10 mg/kg/day doses were next tested, and a dose-dependent increasein H3K4me3 and preservation or restoration of DCX expression inKmt2d^(+/+) or Kmt2d^(+/βGeo) animals in both age groups, respectively,were observed (FIG. 25A, FIG. 25B, FIG. 25C, FIG. 25D and FIG. 24D).This dose also led to a genome-wide increase in H3K4me3 in spleen cellsfrom Kmt2d^(+/βGeo) mice, when compared to Kmt2d^(+/+) littermates onvehicle (FIG. 25E) in association with normalization of expression ofKlf10 (FIG. 26), a known Kmt2d target gene (Guo et al. (2012) Proc.Natl. Acad. Sci. U.S.A. 109:17603-8). In fact, this dose appeared toovercorrect the deficiency (FIG. 25E) which can be observed whenrepresenting data as MA plots (FIG. 27A, FIG. 27B, FIG. 27C, FIG. 27D,and FIG. 27E) or visualizing the shifts in balance among the two states(FIG. 28A, FIG. 28B, FIG. 28C, FIG. 28D and FIG. 28E). Other statecombinations with the same representations were also compared, showing arelative normalization of genome-wide H3K4me3 in Kmt2d^(+/βGeo) micetreated with AR-42, when compared to Kmt2d^(+/+) littermates that did(FIG. 27E) or did not (FIG. 27B) receive drug. The bigger effect atlower intensity Log 2 (CPM) fits with data from ablation ofRubinstein-Taybi gene (CBP) which has dose dose-dependent effects ongene expression thought to depend on the strength of recruitment for aparticular site (Kasper et al. (2010) EMBO J. 29:3660-72).

Improvement of Hippocampal Memory Defects in Kmt2d^(+/βGeo) Mice Treatedwith AR-42:

In keeping with the hypothesis that abnormal GCL neurogenesiscontributes to functional deficits, it was found that performance inhippocampal memory testing correlated with AR-42 dose-dependent effectson DCX expression. Specifically, both Kmt2d^(+/+) and Kmt2d^(+/βGeo)mice showed improved performance on Morris water maze platform crossingduring probe trial (Garthe and Kempermann (2013) Front Neurosci. 7:63)in response to 10 mg/kg/day of AR-42, with a greater response inKmt2d^(+/βGeo) animals and no significant difference between genotypesin the treatment group (FIG. 25F).

Discussion

Prior studies have associated structural abnormalities of the dentategyrus with impaired neurogenesis and hippocampal memory defects (Ansorget al. (2012) BMC Neurosci. 13: 46; Denis-Donini et al. (2008) J.Neurosci. 28:3911-3919). In accordance with the previously observedphenotype in Mll4-targeted mice ((Kerimoglu et al. (2013) J. Neurosci.33, 3452-64), it has been found that heterozygosity for aloss-of-function Kmt2d allele associates a deficiency of H3K4me3 in thedentate gyrus GCL with hippocampal memory defects in a mouse model ofKS. Support for a causal relationship is now greatly enhanced by theobservation disclosed herein that memory deficits can be prevented oreven reversed through systemic delivery of drugs that directly influencethe histone modification events that favor chromatin opening.

The data support the hypothesis that the neurodevelopmental deficiencyin KS is maintained by an impairment of adult neurogenesis due to animbalance between open and closed chromatin states for critical targetgenes. In this light, other Mendelian disorders involving the histonemodification machinery (now numbering over 40; Berdasco and Esteller(2013) Hum. Genet. 132: 359-83) might be amenable to therapeuticintervention with HDAC inhibition (Dash et al. (2009) Neuroscience163:1-8; Vecsey et al. (2007) J. Neurosci. 27: 6128-6140; Gräff and Tsai(2013) Annu. Rev. Pharmacol. Toxicol. 53:311-30). In keeping with thisconcept, neurological phenotypes in mouse models of Rubinstein-Taybisyndrome with haploinsufficiency for the gene encoding the histoneacetyl transferase CREB binding protein (Cbp) respond tointracerebroventricular or intraperitoneal administration of the histonedeacetylase inhibitors SAHA or trichostatin A, respectively (Korzus etal. (2004) Neuron. 42, 961-72; Alarcón et al. Neuron. 42, 947-59 (2004),however no cellular mechanism was described. The specific correlationbetween H3K4me3 and neurogenesis within the dentate gyrus of KS miceoffers a potential unifying mechanism for hippocampal memory defectsseen in inherited defects of the histone modification machinery (Guptaet al. (2010) J. Neurosci. 30:3589-99; Cohen-Armon et al. (2004) Science304:1820-2; Korzus et al. (2004) Neuron. 42:961-72; Alarcón et al.(2004) Neuron. 42:947-59). The further positive correlation of theseevents with functional outcome supports the hypothesis that the fate ofthe GCL in the dentate gyrus is a critical determinant of both diseasepathogenesis and treatment. More work is needed to determine therelative contribution of precursor cell recruitment, differentiation,proliferation and/or survival (Yang et al. (2012) Mol. Cell. Biol.32:3121-31; Lubitz et al. (2007) Mol. Biol. Cell. 18:2356-66). Futurestudies using lineage-specific Kmt2d targeting will help elucidate thecontribution of individual cell populations (GCL, pyramidal layer,molecular layer of the cerebellum) to specific neurodevelopmentalphenotypes.

Although there is an overall decrease in H3K4me3 in the dentate gyrusGCL of Kmt2d^(+/βGeo) mice, substantial cell-to-cell variation is noted.This might reflect redundancy of enzymes capable of adding the H3K4trimethylation mark (Hunter et al. (2012) Nucleic Acids Res. 40: 306-12)that could vary in their expression level (and therefore compensationcapacity) in a cell type- (e.g. differentiation state) or cell state-(e.g. electrochemical environment) dependent manner. Alternatively, thiscould indicate that stochastic events thought to contribute toepigenetic individuality (Bjornsson et al. (2004) Trends Genet. 20:350-8) play a role.

There is precedent that HDACi not only increases histone acetylation,but also H3K4 trimethylation (Huang et al. (2011) Mol. Pharmacol.79:197-206). The presently disclosed indicators nicely illustratecoupling between H4 acetylation and H3K4 trimethylation, withKmt2d^(+/βGeo) mice having defects in both systems. The novel reporteralleles described here have the potential for application in smallmolecule screens to identify drugs with greater potency, specificity andtolerance. There are also many FDA-approved medications, some withlongstanding clinical use, that influence epigenetic modifications inaddition to their originally established functions. An example is theanti-epileptic agent valproic acid, which was recently shown to be apotent HDACi (Phiel et al. (2001) J. Biol. Chem. 276:36734-41). Severalwidely-used supplements or dietary substances, such as folic acid,genestein, and curcumin, are known to influence epigenetic modifications(Meeran et al. (2010) Clin. Epigenetics. 1:101-116). These observationsmay inform the question of potential toxicity of interventions that havebroad effects on pervasive epigenetic events. The apparent tolerance tochronic use of such agents during postnatal life likely reflects, atleast in part, the complex context within which gene transcription andultimate function is achieved. Contributing factors include DNAmodifications, a repertoire of both positive and negative effectors oftranscription, and feedback mechanisms that titrate both gene expressionand protein function. In this light, the predominant influence of agentssuch as HDACi as therapies may prove permissive for correction ofpathologic alterations in physiologic gene expression and functionrather than obligate and therefore less conducive to homeostasis.

Although a beneficial effect of AR-42 treatment on neurogenesis at twodifferent ages (1-2 months and 5-6 months) was demonstrated, suggestingthat this sub-phenotype of KS may be treatable even in adulthood, it iswell established that neurogenesis potential is age-restricted(Martinez-Canabal et al. (2013) Hippocampus 23:66-74). It will beessential to further refine the window of opportunity to influenceneurogenesis in the GCL in both mouse models and patients. It is alsopossible, but as yet unproven, that brief treatment in early postnatalstages will result in the expansion of a stable population of cellswithin the GCL (despite an ongoing relative deficiency ofmethyltransferase function) and hence achieve long-term recovery ofneurologic function. Finally, the ChIP-seq experiments suggest thatAR-42 at a dose of 10 mg/kg/day led to the most improvement infunctional studies (FIG. 25D), but overcorrection of genome-wide H3K4me3(FIG. 25E). Given the favorable tolerance profile of high-dose HDACiwhen used for cancer treatment, this may not be a limiting factor.However, new challenges may arise when HDACi are used chronically for KSor other neurodevelopmental disorders. The combination of in vivoChIP-seq analyses and in vitro reporter allele performance with regardto H3K4me3 status may ultimately allow optimization in the selection ofagent and dose for therapeutic purposes. This concept will be exploredin future work.

Recent advances in Chromatin Immunoprecipitation-Sequencing (ChIP-seq)technologies (Brind'Amour et al. (2015) Nat Commun. 21; 6:6033;Gilfillan G D et al. (2012) BMC Genomics November 21; 13:645) andrelated techniques, such as ATAC-seq (Buenrostro et al. (2015) CurrProtoc Mol Biol. 109:21.29.1-21.29.9), have been performed on cellnumbers that can be practical to extract from patients (500-10,000cells) with a simple blood draw. Accordingly, regarding the Mendeliandisorders of the epigenetic machinery, chromatin immunoprecipitation ofa mark defective in a particular disorder (e.g., H3K4me3 for KS and H4Acfor RTS) followed by some form of quantification (e.g., real time PCR,microarrays, or next generation sequencing) can be used as a diagnostictool or a measurement of therapeutic efficiency. Examples include, butare not limited to, quantifying global levels of H3K4me3 in Kabukisyndrome (expected to be decreased), H3K27me3 in Kabuki syndrome(expected to be increased) and Weaver syndrome (expected to bedecreased), and histone acetylation in Rubinstein-Taybi syndrome(expected to be decreased) and Brachydactyly-Mental Retardation(expected to be increased).

In conclusion, this work suggests that a postnatally ongoing andreversible deficiency of GCL H3K4me3 in association with adultneurogenesis underlies intellectual disability in a mouse model of KS.This work adds to the emerging view that multiple genetic etiologies ofintellectual disability may be amenable to postnatal therapies (Guy etal. (2007) Science 315:1143-7; Das et al. (2013) Sci. Transl. Med. 5:201ra120; Henderson et al. (2012) Sci. Transl. Med. 4:152ra128).

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

That which is claimed:
 1. A method of treating Kabuki syndrome (KS) in apostnatal human subject in need thereof, wherein said subject comprisesa defective KMT2D gene, the method comprising: administering atherapeutically effective amount of an agent that restores balancebetween open and closed chromatin states at one or more target genes,wherein the agent that restores balance between open and closedchromatin states at one or more target genes is an agent thatameliorates the effect of a defective gene encoding a component of theepigenetic machinery, and wherein the agent is(S)-N-hydroxy-4-(3-methyl-2-phenylbutanamido)benzamide (AR-42), whereinsaid administering treats Kabuki syndrome in said subject by crossingthe blood brain barrier.
 2. The method of claim 1, wherein the defectivegene encoding a component of the epigenetic machinery encodes a histonedeacetylase.
 3. The method of claim 1, wherein the defective geneencoding a component of the epigenetic machinery encodes a histonedemethylase.